ISBM - Fall Exam 2

what is the common product of oxidative rxns?

acetyl CoA

what is the term for when increasing levels of ADP and AMP activate glycogen phophorylase and phosphofructokinase-1?

allosteric activators - these regulate allosteric enzymes at the rate-limiting step in the pathway

when is the velocity of an enzyme most sensitive to changes in substrate conc.?

at [S] < Km

how does cAMP activate protein kinase A?

cAMP binds regulatory subunits → dissociation of catalytic subunits → open to bind substrate → goes to town phosphorylating things

what enzyme catalyzes CO₂ + H₂O → H₂CO₃?

carbonic anhydrase CA

what are two catecholamines?

epinephrine and norepinepherine

what is the reverse of glycolysis?

gluconeogenesis

what is the ∆G of the first rxn in the glycolytic pathway? where does the energy come from?

glucose + Pi → glucose-6-P + H₂O → ∆G = 3.3. kcal/mol = energy needed to drive eq. to the right - energy from ATP via substrate level phosphorylation - energy from ↑temp, use of enzyme to ↓act. energy +∆G, Keq < 1.0, product formation not favored → couple w/ ATP + H₂O → ADP + Pi ∆G = - 7.3 kcal/mol → coupled rxn ∆G = -4 kcal/mol

where does regulation occur in glycolysis?

glucose + hexokinase → G6P (-) feedback by G6P *commits glucose to cell cannot go out GLUT receptor! fructose 6 phosphate + PFK-1 → fructose 1,6 bisphosphate (-) feedback by ATP & citrate (+) feedback by AMP *committing rxn for pathway

what happens to glutamate and NH₃ in the presence of ATP?

glutamate + NH₃ + ATP → ADP + Pi + glutamine - coupled rxns that drive otherwise energetically unfavorable rxns - actually two step rxn

what are two polypeptide hormones?

insulin and glucagon

what two hormones determine if fuel is oxidized or stored in the fed state?

insulin and glucagon

pyruvate kinase catalyzes the final step in glycolysis, what are the modifiers of this step?

(+) fructose 1,6 bisphosphate, PEP (substrates) (-) ATP, citrate, acetyl-CoA, protein kinase A (prod. of this driven by epi)

how do drugs and toxins affect enzyme activity?

- the strongest inhibitors are covalent inhibitors, compounds that form covalent bonds with a reactive group in the enzyme active site

when insulin binds to its receptor, what intracellular enzyme stimulates glycogen synthesis?

- PP1 = phophoprotein phosphotase → ↑glycogen synthesis

what are some prostate enzymes that are useful in diagnosis?

- PSA prostate acid phosphatase

what is respiratory quotient?

- RQ = CO₂ produced / O₂ consumed - RQ of glucose = 1.0 [glucose + 6CO₂ → 6O₂ + 6H₂O] - RQ fatty acids = 0.7 - RQ of amino acids ≈ 0.8 - most cells are mixed use, brain prefers glucose (RQ≈1.0) - during fasting, ↓RQ ≈ 0.74

what are Vmax and Km? on what type of curve are they found?

- Vmax = rate of the rxn - Km = [S] that drives enzyme actity at 1/2Vmax = also an indicator of affinity - [S] vs. rate of product formation, hyperbolic

what factors other than blood glucose conc. modulate insulin release?

- autonomic nervous system incl. a branch of the vagus n. - a.a. stimulation - gut hormone release after ingestion of food

what happens to cells following a myocardial infarction?

- MI is caused by atheromatous obstruction or spasm in coronary artery that prevents blood flow to an area of the heart - cells suffer from lack of O2 and fuel - cells cannot generate ATP --> membranes damaged --> enzymes leak from cells - creatine kinase MB (CK or CPK) leak from cells --> can be separated electrophoretically --> amount determinant of whether or not MI occurred - immediately following MI only slight elevation of CK

what is an important cofactor for redox metabolic rxns?

- NAD+ ↔ NADH (freely reversible) → reduced form = NADH → oxidized form = NAD⁺ - FADH₂

are ketoacidic cells starving?

- NO, cells are energy rich, generation of KB in liver from FA used to shuttle acetyl CoA to other areas like skeletal muscle → acetyl CoA → TCA

what is a cofactor? how does that compare to a coenzyme?

- cofactors may influence enzyme activity - cofactors can be metal ions or organic molecules (coenzymes, e.g. NAD, FAD, coenzyme A) - all coenzymes are cofactors but not all cofactors are coenzymes

why are coenzymes classified as cofactors rather than substrates?

- common to so many rxns - original form is regenerated by subsequent rxns - synthesized from vitamins - the amount of coenzyme in the cell is nearly always constant

how does competitive inhibition work?

- competitive inhibitors bind to active site, may or may not be catabolized, regardless they block substrate from binding to active site - ↑Km → ↓affinity - Vmax unchanged

what is a free radical?

- compounds with a free electron, usually in the outer orbital - exist independently in solution or in a lipid environment - many enzymes generate radicals as intermediates in reactions but not usually released into the cell to become free radicals

what are the general properties of amino acids?

- contain: alpha carbon; hydrogen atom; carboxylic acid group (-COOH); amino group (-NH3); side chain (R)

which hormone(s) mobilize fuels during prolonged stress?

- cortisol - activates: a.a. mobilization from muscle, gluconeogenesis (liver & kidney), hormone-sensitive lipase (adipose),

what is the rate limiting enzyme of glycolysis and what does it control?

- PFK-1 is the rate-limiting enzyme of glycolysis and controls the rate of glucose 6P entry into glycolysis - inhibited by ATP - inhibited by citrate + activated by AMP, ADP + activated by fructose 2, 6 bisP made by PFK-2 + norepi --> phosphorylation of PFK2 --> incr. fructose 2,6 bisP --> incr. glycolysis * the most influential modulator of PFK-1 is fructose 2,6 bisphosphate, product of completely separate rxn that also uses fructose 6-phosphate

what is Keq?

- Keq = [P]/[R] - the law, inviolate - e.g. Hb + glucose → HbA1C spontaneous rxn driven by [glucose]

Which type of reactions do each of the following enzymes catalyze? A. Kinase B. Dehydrogenase C. Glycosyltransferase D. Transaminase E. Isomerase

- Kinases transfer phosphate groups - dehydrogenases transfer electrons as hydrogen atoms or hydride ions - Transfer of a carbohydrate residue from one molecule to another is a glycosyltransferase reaction - transaminases transfer amino groups - isomerases transfer atoms within the same molecule.

what is Km?

- Km = [S] that drives enzyme actity at 1/2Vmax - Km is an indicator of affinity,a ↓Km = ↑affinity

how does change in enzyme conc. affect enzyme activity?

- Km same - ↑Vmax with ↑enzyme - ↓Vmax with ↓enzyme

what is phosphofructokinase (PFK)?

- PFK₁ phosphorylates fructose 6 phosphate → F1,6-bisphosphate - as ↑AMP → ↑activity of PFK b/c ↓Km

what are insulin's major actions upon metabolic activity in the body?

(-) glycogenolysis (-) gluconeogenesis (+) glucose uptake in adipose (DNL) and skeletal muscle (TCA) w/ GLUT4 receptors (+) glucose use in TCA (+) glycogen synthesis (+) De Novo Lipogenesis for fat storage →↑glucose (liver) → ↑FA (liver) → VLDL (plasma) → ↑TG in adipose (+) uptake of a.a. in all cells → protein synthesis

what are 3 possible substrates for hexokinase and what are their Km values?

(high affinity) ← glucose Km 0.05 < ATP Km 0.04 < fructose Km 1.5 → (low affinity) - need 400x ATP than glucose, even way more fructose

NORM is eating constantly, what is happening to his levels of liver glucose uptake, liver gluconeogenesis, glycogenolysis and liver glycogen stores?

- (+) net uptake of glucose by the liver → to glycogen → to TG by de novo lipogenesis - liver gluconeogenesis is steady, not zero - liver glycogenolysis is steady, not zero - liver glycogen stores are steady

NORM is now fasting for 12 hrs, what is happening to his levels of liver glucose uptake, liver gluconeogenesis, glycogenolysis and liver glycogen stores?

- (-) net uptake of glucose by the liver → ↓glycogen → ↓TG DNL - liver glycogenolysis↑ → ↑[glucose] - liver gluconeogenesis↑ driven hormonally → ↑[glucose] - ↑insulin - ↑liver uptake of a.a. - liver glycogen↓

how is the insulin receptor a different mechanism of phosphorylation (vs. G protein)?

- (2) insulin bind to α subunits (extracellular) → (2) β transmembrane subunits ∆ conformation → autophosphorylation at tyrosine kinase domain → phosphorylation of target protein

what is the structure of hemoglobin?

- (2) α and (2) β subunits

the finals steps in glycolysis generate energy. using a reversible kinase, then dehydration rxn, then a second substrate level phosphorylation rxn - what enzymes drive these?

- 1,3 BPG + ADP ↔ phosphoglycerate kinase ↔ 3-PG + ATP in first substrate level phosphorylation - 2-PG ↔ enolase/mutase ↔ PEP + H₂O during dehydration rxn - PEP + ADP ↔ pyruvate kinase, cofactors Mg⁺², K⁺ ↔ pyruvate + ATP in second substrate level phosphorylation

what are the 1°, 2°, 3°, 4° structures of proteins?

- 1° sequence of amino acids - 2° α-helices & β-pleating (possible b/c of amide planes) - 3° folding, side chain and hydrophobic interactions - 4° multiple polypeptides

what is amyloidosis?

- AL is when immunoglobulin chains form an insoluble protein aggregate called amyloid in organs and tissues - a conformational change - e.g. Alzheimer disease and familial amyloid polyneuropathy

what are some liver enzymes that are useful in diagnosis?

- ALT (alanine aminotransferase) in hepatocytes for hepatocellular disease - ALP (alkaline phosphatase) in hepatobiliary tree for cholestatic disease - AST (aspartate aminotransferase) in hepatocytes for hepatocellular disease

what are the Bronnsted definitions of an acid and a base?

- An acid is a species having a tendency to lose a proton. - A base is a species having a tendency to accept a proton.

how can one become poisoned by drinking too much alcohol?

- At low concentrations of ethanol, liver alcohol dehydrogenase is the major route of ethanol oxidation to acetaldehyde, a highly toxic chemical. Acetaldehyde not only damages the liver, it can enter the blood and potentially damage the heart and other tissues. - At low ethanol intakes, much of the acetaldehyde produced is safely oxidized to acetate in the liver by acetaldehyde dehydrogenases.

which protein can be used as a marker for chronic inflammation?

- C-reactive protein

which enzymes will confirm an MI?

- CK-MB - LDH 1&2 - troponins I or T (prolonged presence, ↓prognastic indicator) - EKG, CXR, PT, PTT, Brain Naturietic Peptide

why do we get more energy from FA over carbs and AAs?

- FA are more highly reduced, more saturated

what are some important coenzymes?

- Flavin Adenine Dinucleotide ← riboflavin - Nicotinamide Adenine Dinucleotide ← niacin - coenzyme A

how do G proteins regulate through conformational changes?

- G proteins bind GTP --> conformation change --> bind target protein which is either activated or deactivated --> G protein hydrolyzes GTP --> conformation change again and dissociation with target protein

what are the forces that maintain protein 3-D configuration?

- H bonds, salt bridges, disulfide bridges, hydrophobic interactions - folding of protein due to side chain interactions, not random, but is spontaneous and favor a stable structure

where are insulin and glucagon produced?

- Insulin in β-cells of pancreas - Glucagon in α-cells of pancreas

in hormone receptor interactions, what is Ka and Kd?

- Ka = association constant that exists as long as any conc. of constituents is present = [HR]/[H]x[R] - Kd = dissociation constant = [H]x[R]/[HR]

what does Ka indicate?

- Ka is the association constant for the binding of a ligand with a protein - the tighter the binding of a ligand to the protein, the higher the Ka - describes the affinity of a receptor for different drugs

why is histidine an important amino acid?

- a (positive) basic a.a. - has a N-containing ring for a side chain - pKa1 = 1.8; pKaR = 6.0; pKa2 = 9.3 - at low pH all groups carry protons; amino groups have + charge and caroxylic group has 0 charge - as pH increases the proton dissociates from the carboxylic acid group and its charge changes from 0 to -1 (pKa=1.8 the pH at which 50% of the protons have dissociated) - as pH reaches 6 side chain loses proton becomes uncharged - amino group titrates at a higher pH charge changes from +1 to 0 - has a pKa that can donate and accept a proton at a neutral pH, often participates in acid-base catalysis ** offers buffering capacity around pH of 6; ** pI (no net charge) ~pH=7.4

how can a reversible inhibitor be defined?

- a compound that decreases the velocity of the reaction by binding to the enzyme - it is reversible if it is not covalently bound to the enzyme and can dissociate at a significant rate - classified as competitive, noncompetitive, or uncompetitive with respect to their relationship to a substrate - in most rxns the product is a reversible inhibitor

how does a pure noncompetitive inhibitor affect the Vmax and Km of another substrate?

- a noncompetitive inhibitor does not compete with a substrate for its binding site - an increase in substrate will not prevent the inhibitor from binding the allosteric site - the inhibitor in effect lowers the conc. of the active enzyme and therefore lowers the Vmax - if the inhibitor has no effect on the binding site of the substrate, it will not change the Km

what is the role of synthetases?

- a type of ligase that synthesizes bonds in rxns coupled to the cleavage of high-energy phosphate bonds in ATP or other molecules

what is acetylcholinesterase?

- a ubiquitous enzyme - renders acetylcholine into two inert molecules: acetylcholine → choline + acetate → Ach + Ach-ase have covalent and electrostatic bonds → choline reduced and leaves → acetylated enzyme no longer active → hydrolysis → regen of Ach-ase - most effective at high pH - maybe related to histidine??

what is the primary driver of net activity in all cells?

- adenylate charge - as ↓AC → ↑replenishing catabolic exergonic oxidative rxns - as ↑AC → ↑utilizing anabolic endergonic reductive rxns

what is adenylate charge? at what levels are ATP maintained?

- adenylate charge = (ATP + 1/2 ADP) / (ATP + ADP + AMP) - ATP is not the only energy currency in cell but it is high in conc. and maintained that way in a lot of cells!

how is NADH continuously reoxidized back to NAD+ to accept an electron during glycolysis?

- aerobic involves shuttles that transfer reducing equivalents across the mitochondrial membrane - anaerobic when NADH is reoxidized in the cytosol by lactate dehydrogenase (LDH) which reduced pyruvate to lactate

why is it important to keep glucose levels constant? at homeostasis?

- all cells use it - brain prefers it - RBCs use it exclusively

when are ketone bodies produced?

- all they time, used as fuel, important - elevated when ↑[acetyl CoA] and ↑adenylate charge???

what are the two regular secondary structures and how are they maintained?

- alpha-helices and beta-pleated sheets are repeating elements formed by hydrogen bonding between atoms of the peptide bonds

what fuel is used in gluconeogenesis? where does it occur?

- amino acids, lactate oxidation - liver & kidney - amino acids deaminated → urea

what are some pancreatic enzymes that are useful in diagnosis?

- amylase & lipase

what is the role of adenylyl cyclase?

- an enzyme that catalyzes the synthesis of intracellular cyclic adenosine monophosphate (cAMP) - AC has two transmembrane portions along with two intracellular invariant regions that participate in the catalytic activity of cAMP production **at least nine isoforms coded by different genes in different tissues - help cells respond differently to the same hormone!

what is a protein kinase? what is its action?

- an enzyme that transfers a phosphate group from ATP to a protein - phosphorylation introduces a large, bulky, negatively charged group that can alter the activity of a protein - e.g. some adenylyl cyclase (AC) contain serine residues on the intracellular portion that can by phosphorylated by PK

how does temperature affect reaction rates?

- as temp increases the rate of the rxn increases until denaturation occurs

how does phosphorylase function?

- as ↑[AMP] & unphosphorylated + ATP → ↑rate of rxn: glycogen (Glu)x + glycogen phosphorylase → (Glu)x-₁ - as ↑[AMP] & unphosphorylated → ↑↑rxn: glycogen (Glu)x + glycogen phosphorylase → (Glu)x-₁ - as ↑[AMP] & phosphorylated + ATP → ↑↑↑rate of rxn: glycogen (Glu)x + glycogen phosphorylase → (Glu)x-₁ - as ↑[AMP] & phosphorylated → ↑↑↑↑rate of rxn: glycogen (Glu)x + glycogen phosphorylase → (Glu)x-₁

glycogen phosphorylase is endogenously regulated by AMP, ATP & [G6P]. how do insulin and glucagon show exogenous conflicting actions on glycogen phosphorylase?

- as ↑glucagon → ↑[cAMP] → ↑phosphorylation of phosphorylase kinase by PKA → ↑phosphorylation of phosphorylase b → phosphorylase a - as ↑insulin → ↓phosphorylation of phosphorylase kinase by PP1 → ↑dephosphorylation of phosphorylase a → phosphorylase b

how can changes in the rate of a metabolic pathway occur?

- at least one enzyme, the regulatory enzyme has been: --> activated --> inhibited --> increased in conc. --> decreased in conc.

the pKa of the carboxylic acid groups for all amino acids is approximately 2 (1.8-2.4). what are the characteristics of the carboxylic group at pH values lower than pKa, at pH=pKa, and at physiologic pH of 7.4?

- at pH << pKa all the carboxylic acid groups are protonated - when pH = pKa, 50% of the molecues are dissociated into carboxylate anions and protons - at pH = 7.4 most molecules are dissociated and negatively charged

the pKa of the amino groups is approximately 9.5 (8.8-11.0). what form will the amino group be in at physiologic pH of 7.4?

- at pH = 7.4 most amino groups fully protonated and carry positive charge

what is myasthenia gravis?

- autoimmune where Ab act as competitive inhibitors to Ach for Ach-ase - present with fatigue and ↓muscle tone - Dx with tensilon test (edrophonium) - ↑Km, no change to Vmax

how can competitive inhibition be overcome? what effect do they have on Km and Vmax?

- because a competitive inhibitor is similar in structure to the substrate it fits into the active binding site - can overcome the competition by increasing substrate conc. - therefore competitive inhibitors increase the apparent Km of an enzyme because they raise the conc. of substrate necessary to saturate the enzyme - they have no effect of Vmax

how do dehydrogenases act?

- belong to a subclass of oxidoreductases - transfer hydrogen (H atoms or hydride ions) from substrate to an electron-accepting coenzyme such as NAD+

what are glucagon's metabolic influences?

- bind to adiopse and liver cells, some adrenal - ↑lipolysis → ↑fFA → ↑β-oxidation → ↑ketoacidosis - ↑glycogenolysis → ↑blood glucose - ↑uptake of amino acids - ↑gluconeogeneis → nitrogen excrection - ↓glycogenesis - ↓glycolysis? - ↑plasma glucose - ↑ketoacids

what is the secondary messenger mechanism for how the binding of glucagon to its receptor inhibits glycolysis and stimulates gluconeogenesis?

- binding of glucagon → ↑cAMP → protein kinase A → ↑phosphorylation → of PFK2 (↓glycolysis) → of FBPase-2 (↑gluconeogensis) - [fructose 2,6 bisphosphate] affects the stim and inhib as well

how does cholera toxin affect intestinal epithelial cells? what is the treatment?

- binds irreversibly to G protein on cell surface - catalyzes ADP rxn that increases AC activity and thus cAMP levels - normal absorption is diminished - stimulated secretion of Cl- accompanied by cations and water from bloodstream into lumen of gut --> solute-rich diarrhea - Na+ transporters for glucose and a.a. are not affected therefore coadministration of glucose and Na+ by mouth results in uptake of glucose and Na+ accompanied by Cl- and water

how does penicillin work?

- binds tightly to glycopeptidyl transferase/glycopeptide transpeptidase, an enzyme required by bacteria for synthesis of the cell wall by cross-linking components - this is an irreversible inhibitor in the active site = suicide inhibitors

how does ang. II ↑BP by vasoconstriction? what intracellular pathway does it trigger when it binds to GQ protein?

- binds to receptor → α subunit drops GDP picks up GTP → dissociated from βγ →→ IP₃ released → ↑Ca⁺² from ER → binds to calmodulin → activates protein kinase → protein kinase-Ca⁺²CaM phosphorylates stuff →→ DG & Ca⁺² bind to and activate protein kinase C which also phosphorylates stuff

how do the hormones epinephrine (released during stress and exercise) and glucagon (released during fasting) have similar effects?

- both activate synthesis of cAMP --> cAMP activates protein kinase A --> PKA phosphorylates other enzymes, e.g. activate glycogen degradation while inhibiting glycogen synthesis, while incr. release of fatty acid from adipose tissue

how is glucose metabolized in the brain and neural tissues?

- brain and neural tissues very dependent on glucose for energy needs - they generally oxidize glucose via glycolysis and the TCA cycle - except under conditions of starvation glucose is their only major fuel --> roughly 150 g/day - glucose is major precursor of neurotransmitters - as blood glucose drops --> dizzy, light-headed --> comatose and death

how can the binding affinity of a protein for a ligand be quantitatively described?

- by the association constant, Ka, which is the equilibrium constant for the binding reaction of a ligand (L) with a protein (P) - L + P = LP where k1 is the rate constant for association and k2 is the dissociation rate constant of the LP - Keq = k1/k2 = [LP]/[L][P] = Ka = 1/Kd

how is the optimal pH for an enzyme determined?

- by the pKa of the functional groups in the active site

what are some common second messengers? of what are they a product?

- cAMP from adenylate cyclase - cGMP from guanylate cyclase (related to cardiovascular regulation) - diacylglycerol (DAG) & inositol triphosphate (IP₃) ← products of phospholipase C (gen. from intermembrane lipases) - Ca⁺² - measure and sometimes dramatic ∆ in cytoplasm conc.

how does protein kinase A provide a means for hormones to control metabolic pathways? what is the hormonal second messenger in this scenario?

- cAMP is a hormonal second messenger - epi incr. cAMP --> cAMP binds to and activates regulatory subunits of protein kinase A --> which dissociate and --> release the activated catalytic subunits --> which phosphorylates and activates glycogen phosphorylase kinase --> which phosphorylates and activates glycogen phosphorylase **this is a phosphorylation cascase

how can the effect of cAMP be prolonged?

- caffeine inhibits phosphodiesterase which prolongs cAMP existence/activity → continues stimulatory effects

there are LOTS of plasma proteins, what is their role? what do they all have in common?

- can act as buffers - osmotically active, particularly albumin family b/c they are non-specific associators

how are enzyme concentrations affected?

- can be regulated by changes in the rate of enzyme synthesis, i.e. gene transcription - or the rate of degradation

which biomolecules are the three major anionic sustituents? what charge do they carry? what in their name implies this?

- carboxylate groups (-C=O)O-, phosphate groups (-PO4-2), sulfate groups (-SO4-) - the -ate suffix denotes a negative charge

what is the source of ketone bodies?

- catabolism of fatty acids → β-oxidation (liver) → acetyl CoA → acetoacetate & β-hydroxybutyrate → ketone bodies → plasma → uptake by muscle → acetoacetate & β-hydroxybutyrate → acetyl CoA

what factors regulate the secretion of glucagon?

- circulating levels of glucose and insulin - hormone stimulation including epi and cortisol - a.a. stimulation

what are cofactors? how do they affect the active site on an enzyme? where do they come from in the human diet?

- cofactors are metals or complex organic molecules called coenzymes - as substrate binds it might form a covalent intermediate with the cofactor inducing a conformational change that promotes further intereactions - synthesized from vitamins

what are the trace metals in the diet?

- cofactors important to enzymatic activity - e.g. Fe, Cu, Zn, Mg, Mn, K, Ni, Mo, Se

what are cortisol's major actions upon metabolic activity in the body?

- cortisol is a glucocorticoid - affects many tissues by ∆ expression of enzymes (not phosphorylation!) - actions: (+) ↑lipolysis (hormone sensitive lipase); ↑protein degradation (muscle) → ↑free a.a; ↑gluconeogenesis (in liver if ↑precursors, ↑PEPCK); ↑glycogen storage (liver) (-) ↓glucose utilization (adipose & muscle); ↓protein synth (muscle)

what does glucagon signal to occur?

- counterregulatory hormone to insulin - decreased in response to carb meal, elevated during fasting - its conc. in the blood incr. as glucose falls --> promotes glucose production via glycogenolysis --> and gluconeogenesis - I < G --> mobilization of fatty acids from adipose tissue

how do covalent inhibitors work?

- covalent inhibitors form covalent or extremely tight bonds with functional groups in the active catalytic site - e.g. drugs and toxins like DFP an organophosphorus compound prototype for development of sarin gas and insecticides - DFP forms a covalent intermediate in the active site of Ach-ase thereby preventing the enzyme from degrading the neurotransmitter Ach - this inhibition by DFP is essentially irreversible and activity can only be recovered as new enzyme is synthesized

what are some factors that regulate enzymes?

- covalent phosphorylation → may promote (+) or inhibit (-) - non-covalent → conformational ∆ due to allosteric influence, may promote (+) or inhibit (-)

what are some muscle enzymes that are useful in diagnosis?

- creatine kinase (CK), present in many tissues, reversible phosphorylation of creatine: CK-MM (muscle): CK-MB (cardiac); CK-BB (brain, SM of GI, urinary tract) - creatine ↔ creatine-P by CK and ATP or ADP - lactate dehydrogenase (LDH): LDH1&2 myocardium, RBCs, brain; LDH3 brain, kidney; LDH4 liver, kidney, skeletal muscle, brain; LDH5 liver, kidney, skeletal muscle

what is catabolism?

- degradative metabolism involving the release of energy and resulting in the breakdown of complex materials (as carbs, proteins, lipids) - oxidative metabolism, heat producing

what are the symptoms of vitamin deficiencies and what can cause them?

- dietary deficiency is when vitamin coenzyme cofactors def. lead to loss of specific enzyme activities - functional deficiency is from drugs and toxins that inhibit proteins required for coenzyme synthesis, e.g. vitamin transport proteins or biosynthetic enzymes

how can counterregulation of opposing pathways work at the same time to reach the same effect?

- different regulatory enzymes, one activated while the other is inhibited - e.g. glycogen synthesis activated while glycogen degradation inhibited

what is the fate of carbohydrates following a meal?

- digested into monosaccharides (e.g. glucose) - absorbed through intestinal epithelium into the blood --> hepatic portal vein - glucose is: --- oxidized by tissues for energy; --- enters biosynthetic pathways; --- stored as glycogen mainly in liver and muscle - in the liver glucose is converted to triacylglycerols (TG) --> VLDL --> blood --> stored in adiopose or used by cells

what it the fate of dietary protein?

- digested to a.a. --> blood stream --> into cells --> used for protein synthesis or neurotransmitters and heme production - carbon skeleton may be oxidized for energy directly or be converted to glucose

why don't enzymes always fit Michaelis-Menten model?

- does not account for increasing rate due to increasing enzyme conc. - most enzymes have more than one substrate and substrate-binding sites overlap in the catalytic (active) site

how does [glucose] change following a meal? how to I and G change as well?

- driven by hormonal changes - meal consumed → immediate ↑[glucose] → ↓[glucose] and undershoot → return to nonconsumptive levels - [Insulin] pattern follows glucose but lags behind → facilitates glucose uptake at GLUT4 receptors of fat and skeletal muscle cells - [Glucagon] tends to move in opposite direction as I → receptors on liver, adipose and certain kidney cells - I/G ration is the important value

what is an example of a protein-protein interaction in enzyme regulation?

- enzyme activity can be modulated through the reversible binding of a modulator protein - e.g. monomeric G proteins (GTP-binding proteins) activate target proteins through reversible binding

what is covalent modification?

- enzyme activity may also be regulated by covalent modification such as phosphorylation (of a serine, threonine, or tyrosine residue) by a protein kinase

how does substrate concentration affect the rate of enzyme activity?

- enzyme activity: --> incr. with increasing substrate conc. [S] --> reaches max velocity (Vmax) when the enzyme is saturated with substrate - described by Michaelis-Menten eq: vi/Vmax = [S]/[S]+[Km] --> vi = the initial velocity of the rxn --> [S] --> Vmax --> Km = substrate conc. at which vi = 1/2 Vmax

how can the amount of enzyme change?

- enzyme synthesis via protein synthesis, starting with gene expression - regulated protein degradation (during fasting or stress protein degradation in muscle incr. supply of a.a. in blood for gluconeogenesis or synthesis of antibodies, etc - the proteins that target others for degradation are incr. by cortisol)

what are the classifications of the reversible inhibitors?

- enzymes are reversibly inhibited by structural analogs and products - can be: competitive, noncompetitive, uncompetitive

how do enzymes affect Keq? what kinds of ∆G values are "favored"?

- enzymes do not ∆ Keq, they ∆ the rate/ability to achieve Keq - Keq = [P]/[R] +∆G require energy, product formation not favored ↔ Keq < 1.0 -∆G energy released, product formation is favored ↔ Keq > 1.0 ∆G=0 ↔ [prod] = [substrate]

what are allosteric enzymes?

- enzymes where allosteric activators or inhibitors bind to sites other than the active catalytic site and regulate the enzyme through conformational changes that affect the catalytic site

do epinepherine and cortisol work with or as counterregulatory hormones to insulin?

- epi and cortisol have effects on fuel metabolism that oppose insulin, therefore counterregulatory

where in a metabolic pathway do regulatory enzymes occur? why?

- reg. enzymes catalyze the rate-limiting, or slowest, step in the pathway so that incr. or decr. their rate changes the rate of the entire pathway

how does epinephrine promote glycogen degradation → ↑in blood [glucose]?

- epi binds to receptor → ATP→cAMP → activates protein kinase A → activates phosphorylase b kinase → activates glycogen phosphorylase a → glycogen breakdown → glucose 1-phosphate → glucose

epinephrine, glucagon, and ADH all bind to Gs proteins, on what cells does each bind? what is their effector in the cell? and what is the overall effect of each?

- epi: liver & fat cells Gs → adenylyl cyclase → glycogenolysis or lipolysis - glucagon: liver & fat cells Gs → adenylyl cyclase → glycogenolysis or lipolysis - ADH: distal conv. tubule & collecting duct of nephron Gs → adenylyl cyclase → up-reg of aquaporons and water conservation

which hormone(s) mobilize fuels during acute stress?

- epinepherine - activates: glycogenolysis (muscle & liver); lipolysis (adipose)

how are the isozymes of creatine kinase (CK) used clinically?

- exists as tissue-specific isozymes made of two subunits each: MM in skeletal muscle; BB in brain; heart has both MB; two more in mitochondria (heart and universal isoforms) - useful in diagnosing sites of tissue injury and cell death

what are phosphatases?

- family of enzymes responsible for dephosphorylation by hydrolysis but only in presence of phosphatase? - form of covalent modification

what are kinases?

- family of enzymes responsible for hydrolyzing ATP and phosphorylation of something - e.g. protein kinase A - form of covalent modification

what fuel is used in ketone body production? where does it occur?

- fatty acid oxidation (FA → β-oxidation → acetyl CoA → KB) - liver

what do you call the situation when an end product controls the rate of its own synthesis?

- feedback regulation - it usually involves allosteric regulation of the rate-limiting step by the end product of a pathway (or a compound that reflects changes in the conc. of the end product) - the end product may also control its own synth. by inducing or repressing gene transcription but that is slower than allosteric reg.

what metabolic impact does fructose 2,6 bisphosphate have?

- flipping the switch between glycolysis and gluconeogenesis

in addition to providing ATP, what other functions does glycolysis serve?

- generates precursors for biosynthetic pathways --> ribose 5-phosphate --> nucleotides --> other sugars --> glycerol 3 P for TGs --> in liver synthesis of fatty acids

which hormone(s) mobilize fuels during fasting?

- glucagon - activates: glycogenolysis (mostly liver), gluconeogenesis (liver & kidney), hormone-sensitive lipase (adipose)

glucagon and epi have some similar actions, but which types of cells are sensitive to each of these two hormones? which actions are similar?

- glucagon receptors: liver, adipose, some kidney cells (fasting) - epi receptors: α- & β-adrenergic - on liver, adipose, muscle (acute stress) - similar actions: ↑lipolysis, ↑glycogenolysis, ↑gluconeogenesis

how does the Km of the isoenzymes hexokinase and glucokinase compare to each other?

- glucokinase has a much higher Km than other hexokinases - glucokinases are found in the liver & β-cells of pancreas - all other tissue specific hexokinases have Km < 0.2mM

during an extended fast, what process supplies the majority of glucose to the body? does it ramp up quickly or slowly?

- gluconeogenesis (from a.a. & lactate) - begins w/i few hours of last mean, same time as glycogenolysis - slow ratchet up of glucose levels from 4 hrs. post-meal to peak at ~2-3 days

what is the relationship between glucose and triacylglycerols?

- glucose is converted to triacylglycerols - liver packages TG into very low density lipoprotiens (VLDL) --> released into blood - stored as TG in adipose or used by cells

what is the fate of excess glucose in the fed (absorptive) state?

- glucose is major fuel for most tissues - excess glucose stored as glycogen mainly in muscle and liver - excess glucose store as TG in adipose tissue

how is glucose metabolized in red blood cells?

- glucose is the only fuel used by RBCs because they lack mitochondria - glucose is consumed through anaerobic glycolysis which generates ATP and pyruvate --> pyruvate converted to lactate --> lactate released into blood - without glucose, RBCs would not survive!

which cells perform glycolysis and what is generated?

- glucose is the universal fuel for human cells - every human cell type is able to generate ATP from glycolysis where glucose is oxidized and cleaved to form pyruvate - the brain uses glucose almost exclusively as a fuel - RBCs do use glucose exclusively

what is the enzyme that clips off glucose from glycogen?

- glycogen phosphorylase a → active form that cleaves off terminal glucose because it was phosphorylated by "phosphorylase b kinase" - glycogen phosphorylase b → less active form because it was dephosphorylated by "phosphorylase a phosphatase" ↔ allosterically activated by AMP

what is the enzyme responsible for adding glucose to glycogen?

- glycogen synthase a, active when dephosphorylated by phosphoprotein phosphatase - glycogen synthase b, less active when phosphorylated by protein kinase (allosterically stimulated by G6P)

during the initial phase of fasting, what process supplies the majority of glucose to the body? does it ramp up quickly or slowly?

- glycogenolysis within a few hours of last meal - ramps up quickly

why and how is glycolysis regulated?

- glycolysis is regulated to ensure that ATP homeostasis is maintained without using more glucose than is necessary - in most cells hexokinase, the first enzyme, is inhibited by G6P

what is are ketone bodies?

- has the structural group RC(=O)R - Ketone bodies are three water-soluble compounds that are produced as by-products when fatty acids are broken down for energy in the liver and kidney. They are used as a source of energy in the heart and brain. In the brain, they are a vital source of energy during fasting. Although termed "bodies", they are dissolved substances, not particles. - The three ketone bodies are acetone, acetoacetic acid (acetoacetate), and beta-hydroxybutyric acid (beta-hydroxybutyrate), although beta-hydroxybutyric acid is not technically a ketone but a carboxylic acid.

what influences PFK's activity?

- heavily regulated, most complex in glycolytic pathway (+) ↑AMP, ↑ADP, ↑fructose 2,6 bisphosphate* (*most influential modulator though product of totally separate rxn that also uses fructose 6-phosphate) → ↓Km & ↑velocity of rxn (-) ↑ATP, ↑citrate** (**first product of TCA cycle diffuses out of mito to act as neg.modulator) → ↑Km & ↓velocity of rxn

what is hemoglobin? what is its structure?

- hemoglobin is a protein present in RBCs that reversibly binds O2 - adult hemoglobin (HbA) protein comprises four polypeptide chains = 2 alpha + 2 beta

what is the first enzyme in the glycolytic pathway? what is its substrate(s)?

- hexokinase (all cells, RBCs) or glucokinase (liver, β-cells) - glucose & ATP enter active site

how do the tissue distribution, Km, Vmax, substrate specificity, inhibition by G6P compare between hexokinases and glucokinases?

- hexokinase (ubiquitous) Km = 100µM → Vmax lower but operating right at it all the time → all hexoses substrate → inhibited by G6P glucokinase (liver, β-cells) Km = 10µM → acts only when ↑[glucose] → ↑Vmax → D-glucose only substrate → not inhibited by G6P

what does hexokinase do? how does its Km differ in RBCs vs. liver cells?

- hexokinase catalyzes the first step in glucose metabolism in most cells --> it transfers a phosphate from ATP to glucose to form glucose 6-phosphate (G6P) --> hexokinase I in RBCs has Km = 0.05mM; RBCs are totally dependent on glucose metabolism to meet ATP needs and can phosphorylate glucose at rates near Vmax even drastically below the normal fasting level!! --> glucokinase in liver and pancreas has Km = 5-6mM; the rate of phosphorylation of glucose will increase after a high-carb meal and will decrease as blood glucose levels fall ---> the high Km of hepatic glucokinase promotes storage of glucose as liver glycogen or as fat only when glucose is in excess

what is the product inhibitor of hexokinase? why is this beneficial?

- hexokinase is inhibited by glucose 6-phosphate, its product - this helps to conserve blood glucose for tissues that need it

how do blood glucose levels compare between constant eating and intermittent eating?

- higher use and uptake by all cells with constant eating - with intermittent eating get periods of liver glucose export

if epi doesn't go into a cell, how does it have an effect?

- hormone binds to cell surface receptor → 2°messenger produced intracellularly → influence enzymatic activity by phosphorylation - 2° messengers have transient production (de novo) and transient existence (degenerate)

what is the adenylate cyclase system?

- hormone binds to receptor on membrane → activates adenylate cyclase → ↑cAMP → has specific binding sites assoc. w/ protein kinase → activates PK → phosphoylates enzymes (can be + or -) → products

what are the steps in the adenylate cyclase system?

- hormone binds to receptor transmembrane protein coupled w/ Gs protein → conformation ∆ of Gs → GDP dissociates from α subunit → GTP binds to α subunit → α subunit dissociates from β-γ subunits → α subunit assoc. w/ adenylate cyclse → ↓Km of AC for ATP → ↑rate of rxn of ATP → cAMP *no ∆ in [ATP] necessary, only have to ↑affinity of enzyme for substrate

how is the rate of a rxn affected by conc. of the enzyme?

- if you double the amount of enzyme, you will double the amount of product per minute at any substrate conc.

what are chaotropic entities? what are some examples?

- impart chaos on molecular structures - urea, mercaptan disrupt H-bonds and disulfide bridges - temp & pH are entities w/i humans

where does gluconeogenesis occur?

- in the liver and renal cortex

how do enzymes act as catalysts?

- increase the rate of chemical reactions (catalytic power 10^6 to 10^14) - decrease the activation energy which is the energy needed to for rxn to occur - specific binding sites specific to substrates - brings them at the right orientation to react - bind reactants/substrate, convert them to products, release the products - enzymes return to original form - regulate the rate of metabolic pathways in the body

what is the effect of increased glucose levels on insulin? what does the insulin trigger?

- increased blood glucose increases insulin release - glucose is taken up by liver and insulin stimulates: --> used to produce ATP in TCA cycle --> stored as glycogen --> conversion to TG --> VLDL --> release to blood stream - insulin increases glucose uptake by muscle --> storeage as glycogen - insulin increases glucose uptake by adipose tissue and storage as TG

what is the main mechanism of action for glucocorticoids?

- induce enzyme expression - enter nucleus, bind to DNA at glucorticoid regulatory elements → ↑enzyme populations (↓others)

how does penicillin inhibit cell wall synthesis?

- inhibits enzyme activity of cell wall building

which hormone(s) promote fuel storage?

- insulin - activates: glucose storage as glycogen (muscle & liver); stims FA synthesis & storage as TG post high carb meal; stims a.a. uptake and protein synthesis

what happens to intracellular signalling when insulin binds to its receptor?

- insulin binds to extracellular α subunit → ↑ATPase activity → adds phosphate to intracellular tyrosine residues on transmembrane β subunits → array of ???

how is glucose metabolized in adipose tissue?

- insulin stimulates the transport of glucose into adipose cells (as well as muscle) - adipocytes oxidize glucose for energy - they also convert glucose --> TG --> TG stores

how is glucose metabolized in muscle tissue?

- insulin stimulates the transport of glucose into muscle from blood - generate glucose from their own glycogen stores --> can convert glucose to lactate through glycolysis --> can oxidize glucose to CO2 and H2O via TCA - use other fuels such as fatty acids

how does DFP (organophosphate) insecticide and sarin gas work?

- irreversible competitive inhibitor to Ach for Ach-ase * there is an antidote if used quickly to break off O-P group, part of war kits

how is enzymatic activity regulated?

- irreversibly: by proteolytic cleavage - turns on activity like a light switch, e.g. digestive & clotting enzymes - covalently (all the power of regulation): phosphorylation, acetylation, adenylation - non-covalently (freely reversible due to electrostatic attractions): allosteric modification - combo

what do the RBCs do with their glucose?

- lack mitochondria ∴ glycoslysis & lactic acid production (anaerobic resp.) → lactate → diffuses to plasma → liver → gluconeogenesis [Cori Cycle]

what is the carbon skeleton source for gluconeogenesis?

- lactate, amino acids - amino acids deaminated → urea

Two different ligands (A and B) bind to the same receptor on the cell surface. The Kd for ligand A is 10-7M; for ligand B it is 10-9M. Which ligand has the higher affinity for the receptor?

- ligand B has a higher affinity The association constant (Ka) is equal to the reciprocal of the dissociation constant (Kd). The Ka for ligand A is 107M-1, whereas for ligand B it is 109M-1. Ligand B has the higher affinity for the receptor, by 100-fold as compared to ligand A.

during fasting, RBCs continue to exclusively use glucose. from where does it come?

- liver via gluconeogenesis from a.a. and lactate

what are the models for how enzymes and substrates bind to each other?

- lock & key - induced fit - E + S ↔ E-S complex

what are cortisol's metabolic actions on carbohydrate metabolism?

- major influence over glucose use by ∆ing enzyme expression - ↓sensitivity of adipose and muscle cells to inuslin - ↑muscle breakdown - ↑hormone sensitive lipase - ↑gluconeogenesis

how can you improve tissue localization?

- measure activity of other enzymes - measure isozymes * conduct serial measurements

how to myoglobin and hemoglobin compare?

- members of the same globin family - myoglobin is: present in most cells; stores and transports O2 to mitochondria; single polypeptide chain; contains one heme oxygen-binding site - hemoglobin is: present in RBCs; transports O2 from lungs to tissues via blood; composed of four globin chains; four heme oxygen-binding sites ** beta chains evolved first --> alpha chains evolved from duplication of beta chains --> myoglobin assumed to have evolved from gene duplication of the alpha chain *** all three proteins have 15 invariant (identical) residues present

what is so important about fructose 2,6 bisphosphate?

- most influential (+) allosteric modulator on PFK-1 - product of completely different rxn that also uses same substrate (fructose 6-phosphate) - can alleviate the inhibitory effect of ATP

what is the role of alcohol dehydrogenase?

- most ingested ethanol is oxidized to acetaldehyde in the liver by alcohol dehydrogenase (ADH) - ethanol + NAD+ <--> acetaldehyde + NADH + H+ - ADH transfers e- from ethanol to NAD+ to generate acetaldehyde and NADH

muscle glycogen phosphorylase is the rate-limiting enzyme in the pathway of glycogen degradation (glycogen --> glucose-1-phosphate). what regulates this pathway?

- muscle glycogen breakdown is regulated by the allosteric activator AMP which increases in the cell as ATP is used for muscular contraction and more fuel is needed for more ATP generation - it can also be activated through phosphorylation by glycogen phophorylase kinase whose actions increase with incr. epinephrine/adrenaline **both can be active at the same time, especially during exercise

what does a high Km indicate?

- need greater substrate conc. to reach 1/2Vmax - a higher Km indicates decreased rate of substrate binding or decreased enzyme affinity for the substrate

does adenylate cyclase every turn off?

- no, AC always has some activity, always cranking out some cAMP (resting levels) → under influence of receptor G protein ↑activity

how does a non-competitive inhibitor work?

- non-competitive inhibitors bind to allosteric site, substrate binds → product made, but not at same rate - Km same - ↓Vmax

how does glycosylation occur?

- nonenzymatic, the rate is proportional to conc. of glucose present - leads to loss of function and denaturation of the protein, sometimes to a form that cannot be degraded in the cell - glucose in blood, interstitial fluid or ICF binds to exposed amino group - two-step, irreversible process *In HbA1c, the fraction that is usually measured, the glycosylation occurs on an N-terminal valine, glycosylation of hemoglobin has little effect on its function

amino acids are often grouped by the chemical properties of the side chain, what are the groupings and how do they interact with each other and the environment?

- nonpolar hydrophobic a.a.s cluster and exclude water - uncharged polar a.a.s participate in H bonding - cysteine forms disulfide bonds - negatively charged acidic a.a.s form ionic bonds with positively charged molecules such as basic a.a.s

what are the mechanisms by which receptor numbers are subject to change?

- number of receptors in individual is usually steady, may be influenced by pathologic condition - up-regulation: ↑in #of cell-surface receptors; due to prolonged ↓circulating hormonal conc. (or receptor blockade) - down-regulation: ↓# of receptors; due to prolonged ↑circ. hormone e.g. tumor of renal medulla that stims prod. of epi and norepi = pheochromocytoma

how are tissues and organs affected by the ingestion of excessive amounts of alcohol?

- one direct effect is that ethanol is an "antivitamin" that decreases the cellular content of almost every coenzyme - it inhibits the absorption of vitamins and it displaces them from their active/binding sites - e.g. alcoholics develop thiamine deficiency because Etoh inhibits transport of thiamine through intestinal mucosal cells --> oxidation of alpha-keto acids is impaired

what are the benefits of the quaternary structure of proteins?

- opportunity for cooperative binding of ligands (e.g. O2 to hemoglobin) - form binding sites for complex molecules (e.g. antigen binding to Ig) - increased stability (e.g. fibrous collagen proteins) ** prion proteins cause neurodegenerative disease by acting as a template for misfolding

what is the active catalytic site?

- region of the enzyme where the reaction occurs - where functional groups of: coenzymes, tightly bound metals, amino acid residues participate in catalysis

what is the optimal temperature for most human enzymes? how do increases in temp affect enzyme activity?

- optimal temp is ~37 C - increase in temp increases the rate of rxn by increasing vibrational energy of the substrates - further increases in temp cause denaturation due to loss of secondary and tertiary structures

what are the enzyme classes?

- oxidoreductases, transferases, hydrolases, lyases, isomerases, ligases

what are the characteristics of a metabolic pathway regulated at the rate-limiting step?

- pathway regulated at one key enzyme, the regulatory enzyme - this enzyme catalyzes the rate-limiting step - this is the slowest step and is usually not readily reversible - this is usually the first committed step in a pathway

how does Aspirin (acetylsalicylic acid) work?

- pharm drug that exerts its effect through covalent acetylation of an active site in enzyme prostaglandin endoperoxide synthase - aspirin resembles a portion of the prostaglandin precursor that is physiologic substrate for the enzyme

the most heavily regulated, key regulatory, point of no return step in glycolytic pathway is fructose 6-phosphate → fructose 1,6-bisphosphate. what is the enzyme that catalyzes this rxn? what cofactor is necessary? what is the energy source? what is the ∆G?

- phosphofructokinase-1 (PFK-1) - Mg⁺² is a cofactor - ATP → ADP - ∆G ≈ -18 kJ/mol - this is a large value, proceeds viciously to the right

what kind of enzymes regulate other enzyme activity by phosphorylation and dephosphorylation?

- phosphorylation by protein kinase --> the presence of a phosphate group causes alteration in ionic interactions and/or hydrogen bond patterns --> this can make certain enzymes more active and others less active - dephosphorylation by protein phosphatase - works by hydrolysis

which step is considered the first committed step of the glycolysis pathway?

- phosphorylation of fructose 6-P to fructose 1,6-bisphosphate by PFK-1 - this phosphorylation requires ATP and is thermodynamically and kinetically irreversible - PFK-1 irrevocabley commits glucose to the glycolytic pathway - PFK-1 is a regulated enzyme in cells

why is phosphorylation of glucose a committing step?

- phosphorylation of glucose commits it to metabolism within the cell because glucose 6-P cannot be transported back across the plasma membrane - the phosphorylation is irreversible under physiologic conditions because it has a high -delta G' - it does not commit glucose to glycolysis though --> glycolysis, pentose phosphate pathway, glycogen synthesis

what is the first step in glycolysis? where does it occur? what are the end products of glycolysis?

- phosphorylation of glucose to glucose 6-phosphate by hexokinase in RBCs and by glucokinase in liver and pancreatic cells - occurs in the cytosol/cytoplasm - two pyruvate, two NADH, net two ATP

what are some forms of covalent regulatory mechanisms of enzymes?

- phosphorylation, adenylyation, acetylation, methylation ↔ addition of phosphate, adenine, acetyl, methyl onto target residue

what is unique about peptide bonds?

- planar, rigid form - covalent bonds

why are glycolytic intermediates phosphorylated?

- polar and charged → this traps the molecule in the cell, cannot go back out transporter e.g. GLUT - ↓act. energy and ↑specificity of enzymes

what is the structure of insulin?

- polypeptide hormone 51 a.a. long - highly conserved between species - cleaves in three places before secretion --> folds with side chain interactions

what is power of a hormone? what is potency of a hormone?

- power = magnitude of cellular response - potency = magnitude of response to a particular concentration, directly affected by affinity - e.g. ang.II is a powerful vasoconstrictor - e.g. epi is more powerful (↑response) and more potent (cell response at ↓[conc] than norepi

which protein is a useful reflection of amino acid turnover and protein synthesis activity?

- prealbumin (PA) b/c has short half-life

what is important about the pentose phosphate pathway?

- predominant pathway of NADPH production - happens in RBCs - G6P (and other substrates) → by G6P dehydrogenase → ribulose 5-phosphate - G6P dehydrogenase deficit in different ethnic groups

what determines the primary structure of a protein? how can that be modified?

- primary structure is the sequence of amino acids in the protein which is due to DNA sequence coding for each polypeptide - proteins can be modified by phosphorylation, oxidation and carboxylation

what are catecholamines?

- products of the adrenal medulla that are controlled by the nervous system - e.g. epinephrine, isoproterenol, propranolol - β-adrenergic receptor mediated response

what are the three steps of gluconeogenesis?

- pyruvate --> PEP - fructose 1,6-bisP --> fructose 2,6 bisP - glucose 6-P --> glucose

what is the physiological response rate as ligand↑? how much hormone is needed to drive a response?

- quick response to v. small amounts of hormone → ∴levels of hormone cause quick response - do not need a lot of bound receptors to drive phys. response → dramatic ↑w/o reaching Kd

what are the 3 basic types of signal transduction?

- receptor coupling to adenylate cyclase which produces cAMP - receptor kinase activity - receptor coupling to hydrolysis of PIP₂

what are glucagon's major actions upon metabolic activity in the body?

- receptors only on: liver, adipose, certain kidney cells (+) a.a. uptake → glucose ∴ (+) gluconeogenesis (+) glycogenolysis (+) lipolysis by hormone sensitive lipase → free FA in plasma → ↑β-oxidation → acetyl CoA → ↑KB

what influences lactic acid fermentation? why is necessary? what enzyme catalyzes it?

- reduction of pyruvate → lactate is driven by: → ↑[substrate, pyruvate], ↑[cofactor, NADH + H⁺] - catalyzed by lactate dehydrogenase - necessary to regenerate NAD⁺ cofactor - generation of lactate is detrimental, painful, but not a waste product *[lactate] 10x >> [pyruvate]

why is insulin a major anabolic hormone?

- released from beta-cells of the pancreas in response to carbohydrate ingestion - promotes glucose utilization as a fuel for growth and glucose storage as fat and glycogen - increases protein synthesis and cell growth

what is the fate of lactate produced from anaerobic glycolysis?

- released from cells into blood and taken up by other tissues (primarily the liver, heart and skeletal muscle) --> oxidized back to pyruvate --> in liver pyruvate used to synthesize glucose via gluconeogenesis --> glucose returned to blood **The Cori cycle is the cycling of lactate and glucose between peripheral tissues and the liver --> other cells can oxidize lactate to pyruvate for use in the TCA cycle

what do elevated serum creatinine levels indicate?

- renal failure

what is the purpose of the M-M equation?

- represents mathematically the rate of an enzyme-catalyzed reaction that depends on substrate concentration

what is the role of acetylation? what is the role of ADP-ribosylation? what do they have in common?

- reversible acetylation occurs on lysine residues of histones that changes their interaction with phosphate of DNA - ADP-ribosylation is transfer of ADP-ribose from NAD+ to a.a. on target protein in the membrane (primarily leukocytes, skeletal muscle, brain, testes) - this modification may regulate the activity of these proteins

how can exogenous therapeutic factors be used to inhibit enzyme activity?

- reversible: noncovalent binding of a compound w/ enzyme; assoc. w/ active site; allosteric assoc. - irreversible: covalent binding of inhibitor to active site → typically wipes out enzyme's activity, need to regen enzyme to overcome

what are isozymes? what are some examples of diagnostic use?

- same catalytic activity, distinct structure - e.g. Lactate DeHydrogenase (LDH) for pyruvate ↔ lactate is family of enzymes that are different molecular forms with same catalytic activity - M = muscle, H = heart

what cells take up KBs?

- skeletal muscle - NOT RBCs!!

how can cAMP production be slowed?

- slow production of cAMP - make cAMP inactive by phosphodiesterase (there are many and they exist in various cell types)

what are two important principles of hormonal signaling mechanisms that glucagon illustrates?

- specificity of action in tissues is conferred by the receptor on a target cell for glucagon - signal transduction involves amplification of the first message - integration of metabolic responses, i.e. inhibit some, activate others - augmentation and antagonism, e.g. glucagon and epi both incr. cAMP

what promotes catalytic change? how does the rate↑?

- state where free energy is sufficiently large to facilitate change - e.g. CO₂ + H₂O → H₂CO₃ - ↑temp → ↑rate - ↓act. energy → ↑rate

what is meant by the term "reciprocal regulation"?

- stimulating one path, inhibiting another - highly regulated pathways are usually early in steps, e.g. glycolysis phosphorylation of glucose by hexokinase and PFK-1 ???

what is glycogen, where is produced?

- storage form of glucose in animals - short-term means of supplying energy - highly branched polymer of glucose - made in all tissues, predominately in liver and skeletal muscle - 7% weight of liver, 1-2% of muscle (greater mass than liver)

what are antagonists?

- substances which associate/bind with a receptor and promote no response → block epi or others from binding to receptors & initiating phys ∆ - pharmacologic (e.g. propranolol)

what are agonists?

- substances which assoicate/bind with a receptor and ↑specific response → bring about physiologic ∆ - endogenous (e.g. epi) or pharmacologic (e.g. isoproterenol, asthma)

what fits in the active site of an enzyme?

- substrate and potentially competitors

gluconeogenesis is an anabolic process, what is the substrate, what does it produce, where does it occur?

- synthesis of glucose from non-hexose precursors - occurs in all organisms - necessity in mammals as several tissues use glucose as sole or major source of fuel, i.e. RBCs and CNS

what factors affect enzyme activity?

- temp, e.g. cool pre-op patients to slow metabolic activity - pH - [substrate]

how does an enzyme's tertiary shape affect its binding sites?

- the 3-D folding of the protein forms distinct regions called binding sites that are lined with a.a. side chains that interact specifically with another molecule termed a ligand

what is anabolism?

- the constructive part of metabolism concerned especially with macromolecular synthesis - oxidizing agents are reduced, endergonic/energy requiring

what controls if glucose 6-phosphate enters a metabolic pathway? what happens if it does not?

- the control of G6P entry into glycolysis occurs at phosphofructokinase-1 (PFK-1), the rate-limiting enzyme of the pathway - PFK-1 is allosterically inhibited by ATP and allosterically activated by AMP - if G6P does not enter glycolysis, it will inhibit hexokinase

why are shuttles needed for NADH to get from glycolysis to the electron transport chain?

- the inner mitochondrial membrane is impermeable to NADH

what is protein denaturation? what can cause it?

- the loss of the tertiary (and/or secondary) structure within a protein - can be caused by heat, acid, other agents that interfere with H-bonding - usually decreases solubility (precipitation)

what is the relationship between pKa and pH?

- the pKa of an acid is the pH at which it is exactly half dissociated - Clearly, when [AH] = [A-], pH = pKa. - At a pH above the pKa, the acid exists as A- in water, and will therefore be fairly soluble. - At a pH below the pKa, the acid exists mostly as HA in water, and will probably be less soluble.

what determines the 3-D conformation of a protein?

- the primary structure, i.e. the sequence of a.a. - more specifically the sequence of amino acid side chains

how does heavy-metal toxicity of Hg, Pb, Al or Fe occur?

- these metals bind tightly to functional groups of many enzymes - e.g. lead replaces Ca2+ in several regulatory proteins important to the CNS and other tissues

how do pathogenic bacterial toxins like cholera AB toxin, pertussis toxin, and diptheria toxin affect cells?

- they are all ADP-ribosyl tranferases that hydrolyze the N-glycosidic bond on NAD+ and transfer the ADP-ribose portion to a specific a.a. residue on a protein in the affected human cell

besides ATP production, what are other uses for glycolysis in liver and adipose tissue?

- this pathway generates pyruvate as a precursor for fatty acid biosynthesis - provides precursors for the synthesis of compounds such as amino acids and five-carbon sugar phosphates

why make use of plasma enzyme activity?

- to identify the location of cellular/organ damage - to determine the extent of damage - to provide prognostic information as to whether or not therapy is working, improving, stabilizing, etc.

what are some of the functions of proteins in the body?

- transporters of hydrophobic compounds in the blood - cell adhesion molecules that attach cells to each other and to the ECM - hormones that carry signals from one group of cells to another - ion-channels through lipid membranes - enzymes that increase the rate of biochemical reactions

what is the fate of dietary fat?

- triacylglycerols are digested to fatty acids --> in intestinal epithelial cells packaged in chylomicrons and secreted through lymph - stored in adipose cells - oxidized for energy or used in biosynthetic pathway, e.g. membrane lipids

what is another name for triacylglycerols?

- triglycerides or TG

what are some properties of enzymes?

- virtually all proteins - ↑rxn rate - does not affect Keq - not consumed during rxn - specific to reaction type and stereoisomer configuration - enzymes do have half-life, not forever - subject to regulation by: covalent phosphorylation, noncovalent allosteric influences → both may promote (+) or inhibit (-)

what are the regulatory roles of calmodulin (CaM) and Ca⁺²?

- when 4 Ca⁺² bind to calmodulin: →→ smooth muscle contraction →→ exocytosis (e.g. neurotransmitters) →→ activation of cAMP phosphodiesterase

when does anaerobic glycolysis occur? where does it occur? what is produced?

- when oxygen is limited, or there are few or no mitochondria, or under increased demands for ATP - occurs in the cytosol/cytoplasm - lactate dehydrogenase oxidizes NADH generated from glycolysis by reducing pyruvate to lactate - lactate is released into the blood --> under hypoxic conditions can lead to lactic acidemia

if measuring CK due to high suspicion of MI, when and for how long should it be monitored? what else could be monitored as supplemental measurements?

- within 2 hours of the onset of an acute MI, the MB form of CK (reference range of 38 to 174 U/ L) begins leaking from heart cells that were injured by the ischemic process. These rising serum levels of the MB fraction (and, therefore, of the total CK) reach their peak 12 to 36 hours later and usually return to normal within 3 to 5 days from the onset of the infarction (Fig. 6.15). - In addition to the CK measurements, blood can be analyzed for myoglobin and the heart isoform of troponin T (TnT), a protein involved in muscle contraction

what are epinephrine's major actions upon metabolic activity in the body?

- α and β adrenergic receptors on: liver, adipose, muscle (+) lipolysis in adipose tissue → free FA & glycerol in plasma → glycerol → liver → ↑gluconeogenesis → glucose → glycogen → glucose → (+) glycogenolysis in liver → ↑blood glucose (+) glycogenolysis in muscle → ↑ intracellular glucose → ↑pyruvate & lactate → ↑ECF [lactate] → liver → gluconeogenesis

how do allosteric influences affect enzyme activity?

- ↑ or ↓ Km - no change to Vmax - substrate binds, product made but at different rate

as insulin↑, what processes ↑ or ↓?

- ↑glucose use by cells → ↑conversion to glycogen → ↑pentose pathway - ↓plasma conc. of a.a., fFA, glycerol - ↓gluconeogenic activity, but not zero

why does acetaminophen cause liver damage?

- ↓disulfide bridges

which fuels are used during fasting?

- ↓glucose in all cells - ↑a.a. uptake → ↑gluconeogenesis in liver and kidney - ↑FA → ↑ketoacids (β-hydroxybutyrate & acetoacetate) use

what is the ∆G of the glycolytic pathway?

- ∆ G = -22 kcal/mol - therefore it cannot be reversed without the expenditure of energy

how many hours does it take to deplete liver stores of glycogen?

30 hours, after this gluconeogenesis is the only source of blood glucose

Which of the following shows the linear sequence of atoms joined by covalent bonds in a peptide backbone? A. -N- C- C- N- C- C- N- C- C B. -N- C- O- N- C- O- N- C- O- C. -N- C- C- O- N- C- C- O- N- C- C- O- D. -N- H- C- C- N- H- C- C- N- H- C- C- E. -N- H- C- O- H- N- H- C- O- H- N- H- C- C-

A. -N- C- C- N- C- C- N- C- C The answer is A. The peptide backbone contains only carbon and nitrogen atoms linked covalently in a chain. The amide nitrogen of one amino acid (N) is covalently attached to the α-carbon of the same amino acid (C), which is covalently linked to the carboxyl carbon (C) of that amino acid, which forms a peptide bond with the nitrogen of the next amino acid (N).

Di Abietes's different preparations of insulin contain some insulin complexed with protamine that is absorbed slowly after injection. Protamine is a protein preparation from rainbow trout sperm containing arginine-rich peptides that bind insulin. Which of the following provides the best explanation for complex formation between protamine and insulin? A. Arginine is a basic amino acid that binds to negatively charged amino acid side chains in insulin. B. Arginine is a basic amino acid that binds to the α-carboxylic acid groups at the N-terminals of insulin chains. C. Arginine is a large bulky hydrophobic amino acid that complexes with leucine and phenylalanine in insulin. D. Arginine forms disulfide bonds with the cysteine residues that hold the A and B chains together. E. Arginine has a side chain that forms peptide bonds with the carboxyl terminals of the insulin chains.

A. Arginine is a basic amino acid that binds to negatively charged amino acid side chains in insulin. The answer is A. Arginine is a basic amino acid that has a positively charged side chain at neutral pH. It can, therefore, form tight electrostatic bonds with negatively charged asp and glu side chains in insulin. The α- carboxylic acid groups at the N-terminals of proteins are bound through peptide bonds (thus, B is incorrect). The arginine side chain is not hydrophobic, and it cannot form disulfide bonds because it has no sulfhydryl group (thus, C and D are incorrect). Its basic group is an ureido group that cannot form peptide bonds (see E).

A male patient exhibited a BMI of 33 kg/ m2 and a waist circumference of 47 in. What dietary therapy would you consider most helpful? A. Decreased intake of total calories, because all fuels can be converted to adipose tissue triacylglycerols. B. The same amount of total calories, but substitution of carbohydrate calories for fat calories. C. The same amount of total calories, but substitution of protein calories for fat calories. D. A pure-fat diet, because only fatty acids synthesized by the liver can be deposited as adipose triacylglycerols. E. A limited food diet, such as the ice cream and sherry diet.

A. Decreased intake of total calories, because all fuels can be converted to adipose tissue triacylglycerols. The answer is A. The patient's BMI is in the obese range, with large abdominal fat deposits. He needs to decrease his intake of total calories because an excess of calories ingested as carbohydrate, fat, or protein results in deposition of triacylglycerols in adipose tissue. If he keeps his total caloric intake the same, substitution of one type of food for another will help very little with weight loss. (However, a decreased intake of fat may be advisable for other reasons). Limited food diets, such as the ice cream and sherry diet, or a high protein diet of shrimp, work if they decrease appetite and, therefore, ingestion of total calories.

A patient was diagnosed with a hypertriglyceridemia. This condition is named for the high blood levels of lipids composed of which one of the following? A. Three fatty acyl groups attached to a glycerol backbone B. A glycerol lipid containing a phosphorylcholine group C. A sphingolipid containing three fatty acyl groups D. Three glycerol moieties attached to a fatty acid E. Three glyceraldehyde moieties attached to a fatty acid

A. Three fatty acyl groups attached to a glycerol backbone The answer is A. Triglycerides (triacylglycerols) are composed of a glycerol backbone to which three fatty acids (" tri-") are attached (see Fig. 5.18). The carboxylic acid group of each fatty acid forms an ester with one of the three -OH groups on glycerol; thus, the fatty acid is an "acyl" group. Sphingolipids, in contrast, contain one fatty acyl group and one group derived from a fatty acid, attached to a group derived from serine (see Fig. 5.20).

cAMP is the intracellular second messenger for several hormones that regulate fuel metabolism. The specificity of the physiologic response to each hormone results from the presence of specific receptors for that hormone in target tissues. How do these receptors affect adenylate cyclase and cAMP?

Although glucagon works by activating adenylate cyclase, a few hormones inhibit adenylate cyclase. In this case, the inhibitory G-protein complex is Gi-complex. For example, glucagon activates glucose production from glycogen in liver but not in skeletal muscle because glucagon receptors are present in liver but absent in skeletal muscle. However, skeletal muscle has adenylate cyclase, cAMP, and PKA, which can be activated by epinephrine binding to the β2-receptors in the membranes of muscle cells. Liver cells also have epinephrine receptors. cAMP is very rapidly degraded to AMP by a membrane-bound phosphodiesterase. The concentration of cAMP is thus very low in the cell, so changes in its concentration can occur rapidly in response to changes in the rate of synthesis. The amount of cAMP present at any time is a direct reflection of hormone binding and the activity of adenylate cyclase. It is not affected by ATP, ADP (adenosine diphosphate), or AMP levels in the cell. cAMP transmits the hormone signal to the cell by activating PKA (cAMP-dependent protein kinase). As cAMP binds to the regulatory subunits of PKA, these subunits dissociate from the catalytic subunits, which are thereby activated. Activated PKA phosphorylates serine residues of key regulatory enzymes in the pathways of carbohydrate and fat metabolism. Some enzymes are activated and others are inhibited by this change in phosphorylation state. The message of the hormone is terminated by the action of semispecific protein phosphatases that remove phosphate groups from the enzymes. The activity of the protein phosphatases is also controlled through hormonal regulation.

Elevated levels of chylomicrons were measured in the blood of a patient. A dietary therapy, which decreased which one of the following answer choices would be most helpful in lowering chylomicron levels? A. Overall calories B. Fat C. Cholesterol D. Starch E. Sugar

B. Fat Answer is B. Chylomicrons are the lipoprotein particles formed in intestinal epithelial cells from dietary fats, and they contain principally triacylglycerols formed from components of dietary triacylglycerols. A decreased intake of calories in general would include a decreased consumption of fat, carbohydrate, and protein, which might not lower chylomicron levels. Dietary cholesterol, although found in chylomicrons, is not their principal component.

Which of the following characterize α-helix regions of proteins? A. They all have the same primary structure. B. They are formed principally by hydrogen bonds between a carbonyl oxygen atom in one peptide bond and the amide hydrogen from a different peptide bond. C. They are formed principally by hydrogen bonds between a carbonyl atom in one peptide bond and the hydrogen atoms on the side chain of another amino acid. D. They are formed by hydrogen bonding between two adjacent amino acids in the primary sequence. E. They require a high content of proline and glycine.

B. They are formed principally by hydrogen bonds between a carbonyl oxygen atom in one peptide bond and the amide hydrogen from a different peptide bond. The answer is B. The regular repeating structure of an α-helix is possible because it is formed by hydrogen bonds within the peptide backbone of a single strand. Thus, α-helices can be formed from a variety of primary structures. However, proline cannot accommodate the bends for an α-helix because the atoms involved in the peptide backbone are part of a ring structure, and glycine cannot provide the space filling required for a stable structure.

what is the relationship between pKa, Ka, and acid strength?

Because of the minus sign, the lower the pKa, the higher the Ka and the stronger the acid.

After digestion of a high-carbohydrate meal, which one of the following is most likely to occur? A. Glucagon is released from the pancreas. B. Insulin stimulates the transport of glucose into the brain. C. Liver and skeletal muscle use glucose as their major fuel. D. Skeletal muscles convert glucose to fatty acids. E. Red blood cells oxidize glucose to CO2.

C. Liver and skeletal muscle use glucose as their major fuel. The answer is C. After a high-carbohydrate meal, glucose is the major fuel for most tissues, including skeletal muscle, adipose tissue, and liver. The increase in blood glucose levels stimulates the release of insulin, not glucagon. Insulin stimulates the transport of glucose in skeletal muscle and adipose tissue, not brain. Liver, not skeletal muscle, converts glucose to fatty acids. Although the red blood cell uses glucose as its only fuel at all times, it generates ATP from conversion of glucose to lactate, not CO2.

Which of the following is a characteristic of globular proteins? A. Hydrophilic amino acids tend to be on the inside. B. Hydrophobic amino acids tend to be on the outside. C. Tertiary structure is formed by hydrophobic and electrostatic interactions between amino acid side chains and by hydrogen bonds between peptide bonds. D. Secondary structures are formed principally by hydrophobic interactions between amino acids. E. Covalent disulfide bonds are necessary to hold the protein in a rigid conformation.

C. Tertiary structure is formed by hydrophobic and electrostatic interactions between amino acid side chains and by hydrogen bonds between peptide bonds. The answer is C. Globular proteins fold into a spherelike structure with their hydrophilic residues on the outside to interact with water (hence, A is incorrect) and their hydrophobic residues on the inside away from water (thus, B is incorrect). Secondary structures are formed by hydrogen bonding, not hydrophobic bonding (thus, D is incorrect). Disulfide bonds are rare in globular proteins and are not needed to maintain a stable structure (thus, E is incorrect).

Insulin release in the fed state will lead to which of the following metabolic changes?

C. Yes increased glucose transport by muscle; Yes VLDL synthesis by the liver; No FA synth in fat cells; Yes glycogen synthesis in liver The answer is C. In the fed state, insulin will be released because of the increase in blood glucose levels. Insulin will act on muscle cells to increase glucose uptake in the muscle. Insulin will also stimulate the liver to synthesize both glycogen and fatty acids, which leads to enhanced triglyceride synthesis and very low-density lipoprotein (VLDL) production to deliver the fatty acids to other tissues of the body. Insulin will stimulate glucose uptake in fat cells, but does not stimulate fatty acid synthesis in the fat cells (i.e., unique to the liver), but will lead to enhanced triglyceride synthesis in the fat cells.

Which of the following is a universal characteristic of water-soluble organic compounds? A. They are composed of carbon and hydrogen atoms. B. They must contain a group that has a full negative charge. C. They must contain a group that has a full positive charge. D. They contain polar groups that can hydrogen bond with water. E. They contain aromatic groups.

D. They contain polar groups that can hydrogen bond with water. The answer is D. Water-soluble compounds are polar (contain an uneven distribution of charge) so that the more positive portion of the molecule hydrogen-bonds (shares electrons) with the oxygen of water, and the more negative portion of the molecule hydrogen-bonds with the hydrogens of water. Water-soluble compounds need not contain a full negative or positive charge (thus, B and C are incorrect) and generally contain oxygen or nitrogen, in addition to carbon and hydrogen (see A). Large C- H portions of a molecule, such as an aromatic ring, are nonpolar and contribute to the insolubility of the molecule in water (see E).

During digestion of a mixed meal, which one of the following is most likely to occur? A. Starch and other polysaccharides are transported to the liver. B. Proteins are converted to dipeptides, which enter the blood. C. Dietary triacylglycerols are transported in the portal vein to the liver. D. Monosaccharides are transported to adipose tissue via the lymphatic system. E. Glucose levels increase in the blood.

E. Glucose levels increase in the blood. The answer is E. During digestion of a mixed meal, starch and other carbohydrates, proteins, and dietary triacylglycerols are broken into their monomeric units (carbohydrates into simple monosaccharides, protein into amino acids, triacylglycerols into fatty acids and glycerol). Glucose is the principal sugar in dietary carbohydrates, and thus it increases in the blood. Amino acids and monosaccharides enter the portal vein and go to the liver first. After digestion of fats and absorption of the fatty acids, most fatty acids are converted back into triacylglycerols and subsequently into chylomicrons by intestinal cells. Chylomicrons go through lymphatic vessels and then blood, principally to adipose tissue.

what is the only oxidation-reduction rxn in the glycolytic pathway?

G3P + NAD⁺ + HPO₄⁻² ↔ 1,3 BPG + NADH + H⁺ ∆G = -11.8 kcal/mol

As Ann O'Rexia eats a high-carbohydrate meal, her blood glucose will rise to approximately 20 mM in the portal vein, and much of the glucose from her carbohydrate meal will enter the liver. How will the activity of glucokinase in the liver change as glucose is increased from 4 mM to 20 mM? (Hint: Calculate vi as a fraction of Vmax for both conditions, using a Km for glucose of 5 mM and the Michaelis-Menten equation).

Glucokinase, which has a high Km for glucose, phosphorylates glucose to glucose 6-phosphate about twice as fast after a carbohydrate meal as during fasting. Substitute the values for S and Km into the Michaelis-Menten equation. The initial velocity will be 0.44 times Vmax when blood glucose is at 4 mM and about 0.80 times Vmax when blood glucose is at 20 mM. In the liver, glucose 6-phosphate is a precursor for both glycogen and fat synthesis. Thus, these storage pathways are partially regulated through a direct effect of substrate supply. They are also partially regulated through an increase of insulin and a decrease of glucagon, two hormones that signal the supply of dietary fuel.

how does signal transuction by insulin work?

Insulin binds to a receptor on the plasma membrane of target cells. The receptor has two types of subunits: the α-subunits to which insulin binds, and the β-subunits, which span the membrane and protrude into the cytosol. The cytosolic portion of the β-subunit has tyrosine kinase activity. On binding of insulin, the tyrosine kinase phosphorylates tyrosine residues on the β-subunit (autophosphorylation), as well as on several other enzymes within the cytosol. The basic tissue-specific cellular responses to insulin, however, can be grouped into five major categories: (1) insulin reverses glucagon-stimulated phosphorylation of the enzymes of carbohydrate metabolism, (2) insulin works through a phosphorylation cascade that stimulates the phosphorylation of several enzymes, (3) insulin induces and represses the synthesis of specific enzymes, (4) insulin acts as a growth factor and has a general stimulatory effect on protein synthesis, and (5) insulin stimulates glucose and amino acid transport into cells. From the student's point of view, the ability of insulin to reverse glucagon-stimulated phosphorylation occurs as if it were lowering cAMP and stimulating phosphatases that could remove those phosphates added by protein kinase A (PKA). In reality, the mechanism is more complex and still not fully understood.

how do you quantitatively characterize the affinity of a binding site for its ligand?

Ka the association or affinity constant

what are Kd and Bmax?

Kd = [ligand] at 50% receptor occupancy Bmax = total amount of receptor occupancy

what drives a rxn?

Keq =

what controls glycogen synthesis & degradation rxn direction?

Keq = [P]/[R] = G6P/G1P = 19 rxn direction driven by conc. of reactants and products

what is the Km of carbonic anhydrase for HCO₃⁻?

Km 9, middle value and fairly fast rxn

what happens during oxidation and reduction?

Reaction | Electrons | Hydrogen | Oxygen Oxidation | lose e- | lose H | gain O (or OH group) Recuction | gain e- | gain H | lose O

An allosteric enzyme has the following kinetic properties: a Vmax of 25 U/ mg enzyme, and a Km, app of 1.0 mM. These kinetic parameters were then measured in the presence of an allosteric activator. Which one of the following would best describe the findings of that experiment? A. A Vmax of 25 U/ mg enzyme, and a Km, app of 0.2 mM. B. A Vmax of 15 U/ mg enzyme, with a Km, app of 2.0 mM. C. A Vmax of 25 U/ mg enzyme, with a Km, app of 2.0 mM. D. A Vmax of 50 U/ mg enzyme, with a Km, app of 5.0 mM. E. A Vmax of 50 U/ mg enzyme, with a Km, app of 10.0 mM.

The answer is A. Allosteric activators will shift the sigmoidal kinetic curve for the enzyme to the left, thereby reducing the Km, app (so one-half maximal velocity will be reached at a lower substrate concentration), without affecting the maximal velocity (although in some cases Vmax can also be increased). Allosteric inhibitors will shift the curve to the right, increasing the Km, app and, sometimes, also decreasing the Vmax.

The pancreatic glucokinase of a patient with MODY had a mutation replacing a leucine with a proline. The result was that the Km for glucose was decreased from a normal value of 6 mM to a value of 2.2 mM, and the Vmax was changed from 93 U/ mg protein to 0.2 U/ mg protein. Which of the following best describes the patient's glucokinase compared with the normal enzyme? A. The patient's enzyme requires a lower concentration of glucose to reach ½ Vmax. B. The patient's enzyme is faster than the normal enzyme at concentrations of glucose < 2.2 mM. C. The patient's enzyme is faster than the normal enzyme at concentrations of glucose > 2.2 mM. D. At near-saturating glucose concentration, the patient would need 90 to 100 times more enzyme than normal to achieve normal rates of glucose phosphorylation. E. As blood glucose levels increase after a meal from a fasting value of 5 mM to 10 mM, the rate of the patient's enzyme will increase more than the rate of the normal enzyme.

The answer is A. The patient's enzyme has a lower Km than the normal enzyme and, therefore, requires a lower glucose concentration to reach ½ Vmax. Thus, the mutation may have increased the affinity of the enzyme for glucose, but it has greatly decreased the subsequent steps of the reaction leading to formation of the transition-state complex, and thus Vmax is much slower. The difference in Vmax is so great that the patient's enzyme is much slower whether you are above or below its Km for glucose. You can test this by substituting 2 mM glucose and 4 mM glucose into the Michaelis-Menten equation, v = Vmax S/( Km + S) for the patient's enzyme and for the normal enzyme. The values are 0.0095 U/ mg and 0.0129 U/ mg for the patient's enzyme versus 23.2 U/ mg and 37.2 U/ mg for the normal enzyme, respectively (thus, B and C are incorrect). At near-saturating glucose concentrations, both enzymes will be near Vmax, which is equal to kcat times the enzyme concentration. Thus, it will take nearly 500 times as much of the patient's enzyme to achieve the normal rate (93 % 0.2), and so C is incorrect. E is incorrect because rates change most as you decrease substrate concentration below the Km. Thus, the enzyme with the highest Km will show the largest changes in rate.

While studying a novel pathway in a remote species of bacteria, you discover a new globular protein that phosphorylates a substrate, using ATP as the phosphate donor. Which of the following structures most likely contains this protein? A. An actin fold B. An immunoglobulin fold C. A nucleotide-binding fold D. A globin fold E. A β-barrel

The answer is A. The protein hydrolyzes ATP, which is a characteristic of the actin fold. None of the other folds described will hydrolyze ATP.

Lysozyme is an enzyme that cleaves glycosidic linkages in bacterial cell walls. The pH optimum of the purified enzyme is 5.2. There are two acidic residues at the active site of lysozyme (E35 and D52) that are required for enzyme activity. The pKa of E35 is 5.9, whereas the pKa of D52 is 4.5. What are the primary ionization states of these two residues at the pH optimum of the enzyme? A. E35 is protonated, D52 is ionized. B. E35 is protonated, D52 is protonated. C. E35 is ionized, D52 is protonated. D. E35 is ionized, D52 is ionized. E. This cannot be determined from the information provided.

The answer is A. When the pKa of an ionizable group is below the pH value, the group will be deprotonated. When the pKa of an ionizable group is above the pH value, the group will be protonated. Thus, at pH 5.2, glutamate 35 (with a pKa of 5.9, which is greater than 5.2) will remain protonated, and aspartate 52 (with a pKa of 4.5, which is less than 5.2) will be ionized (because the side chain carries a negative charge when deprotonated). Therefore, E35 is protonated, and D52 is ionized. There is sufficient information presented to answer this question.

Methanol (CH3OH) is converted by alcohol dehydrogenases to formaldehyde (CH2O), a compound that is highly toxic to humans. Patients who have ingested toxic levels of methanol are sometimes treated with ethanol (CH3CH2OH) to inhibit methanol oxidation by alcohol dehydrogenase. Which of the following statements provides the best rationale for this treatment? A. Ethanol is a structural analog of methanol and might therefore be an effective noncompetitive inhibitor. B. Ethanol is a structural analog of methanol that can be expected to compete with methanol for its binding site on the enzyme. C. Ethanol can be expected to alter the Vmax of alcohol dehydrogenase for the oxidation of methanol to formaldehyde. D. Ethanol is an effective inhibitor of methanol oxidation regardless of the concentration of methanol. E. Ethanol can be expected to inhibit the enzyme by binding to the formaldehyde-binding site on the enzyme, even though it cannot bind at the substrate-binding site for methanol.

The answer is B. Ethanol has a structure very similar to methanol (a structural analog) and thus can be expected to compete with methanol at its substrate-binding site. This inhibition is competitive with respect to methanol and, therefore, Vmax for methanol will not be altered and ethanol inhibition can be overcome by high concentrations of methanol (thus, A, C, and D are incorrect). E is illogical because the substrate methanol stays in the same binding site as it is converted to its product, formaldehyde.

A patient was born with a congenital mutation in an enzyme that severely affected its ability to bind an activation-transfer coenzyme. As a consequence, which one of the following is most likely to occur? A. The enzyme will be unable to bind the substrate of the reaction. B. The enzyme will be unable to form the transition-state complex. C. The enzyme will normally use a different activation-transfer coenzyme. D. The enzyme will normally substitute the functional group of an active-site amino acid residue for the coenzyme. E. The reaction may be carried out by the free coenzyme, provided the diet carries an adequate amount of its vitamin precursor.

The answer is B. In most reactions, the substrate binds to the enzyme before its reaction with the coenzyme occurs. Thus, the substrate may bind but it cannot react with the coenzyme to form the transition-state complex.

Which of the following describes a characteristic feature of an enzyme that obeys Michaelis-Menten kinetics? A. The enzyme velocity is at one-half the maximal rate when 100% of the enzyme molecules contain bound substrate. B. The enzyme velocity is at one-half the maximal rate when 50% of the enzyme molecules contain bound substrate. C. The enzyme velocity is at its maximal rate when 50% of the enzyme molecules contain bound substrate. D. The enzyme velocity is at its maximal rate when all of the substrate molecules in solution are bound by the enzyme. E. The velocity of the reaction is independent of the concentration of enzyme.

The answer is B. The rate of the reaction is directly proportional to the proportion of enzyme molecules that contain bound substrate. Thus, it is at 50% of its maximal rate when 50% of the molecules contain bound substrate (thus, A, C, and D are incorrect). The rate of the reaction is directly proportional to the amount of enzyme present, which is incorporated into the term Vmax (where Vmax = k[ total enzyme]) (thus, E is incorrect).

A rate-limiting enzyme catalyzes the first step in the conversion of a toxic metabolite to a urinary excretion product. Which of the following mechanisms for regulating this enzyme would provide the most protection to the body? A. The product of the pathway should be an allosteric inhibitor of the rate-limiting enzyme. B. The product of the pathway should act through gene transcription to decrease synthesis of the enzyme. C. The toxin should act through gene transcription to increase synthesis of the enzyme. D. The enzyme should have a high Km for the toxin. E. The toxin allosterically activates the last enzyme in the pathway.

The answer is C. The most effective regulation should be a feed-forward type of regulation in which the toxin activates the pathway. One of the most common ways this occurs is through the toxin acting to increase the amount of enzyme by increasing transcription of its gene. A and B describe mechanisms of feedback regulation, in which the end product of the pathway decreases its own rate of synthesis and are, therefore, incorrect. D is incorrect because a high Km for the toxin might prevent the enzyme from working effectively at low toxin concentrations, although it would allow the enzyme to respond to increases of toxin concentration. It would do little good for the toxin to allosterically activate any enzyme but the rate-limiting enzyme (thus, E is incorrect).

An individual had a congenital mutation in glucokinase in which a proline was substituted for a leucine on a surface helix far from the active site but within the hinge region of the actin fold. This mutation would be expected to have which one of the following effects? A. It would have no effect on the rate of the reaction because it is not in the active site. B. It would have no effect on the rate of the reaction because proline and leucine are both nonpolar amino acids. C. It would have no effect on the number of substrate molecules reaching the transition state. D. It would probably affect the binding of ATP or a subsequent step in the reaction sequence. E. It would probably cause the reaction to proceed through an alternate mechanism.

The answer is D. The patient was diagnosed with maturity-onset diabetes of the young (MODY) caused by this mutation. In glucokinase, binding of glucose normally causes a huge conformational change in the actin fold that creates the binding site for ATP. Although proline and leucine are both nonpolar amino acids, B is incorrect— proline creates kinks in helices and thus would be expected to disturb the large conformational change required (see Chapter 7). In general, binding of the first substrate to an enzyme creates conformational changes that increases the binding of the second substrate or brings functional groups into position for further steps in the reaction. Thus, a mutation need not be in the active site to impair the reaction, and A is incorrect. It would probably take more energy to fold the enzyme into the form required for the transition-state complex, and fewer molecules would acquire the energy necessary (thus, C is incorrect). The active site lacks the functional groups required for an alternate mechanism (thus, E is incorrect).

what affects the amount of CAMP present at any time in a cell?

The concentration of cAMP is thus very low in the cell, so changes in its concentration can occur rapidly in response to changes in the rate of synthesis. The amount of cAMP present at any time is a direct reflection of hormone binding and the activity of adenylate cyclase. It is not affected by ATP, ADP (adenosine diphosphate), or AMP levels in the cell. cAMP transmits the hormone signal to the cell by activating PKA (cAMP-dependent protein kinase). As cAMP binds to the regulatory subunits of PKA, these subunits dissociate from the catalytic subunits, which are thereby activated. Activated PKA phosphorylates serine residues of key regulatory enzymes in the pathways of carbohydrate and fat metabolism. Some enzymes are activated and others are inhibited by this change in phosphorylation state. The message of the hormone is terminated by the action of semispecific protein phosphatases that remove phosphate groups from the enzymes. The activity of the protein phosphatases is also controlled through hormonal regulation.

are the Km of the alcohol dehydrogenases low or high? how does alcoholism lead to liver damage?

The liver alcohol dehydrogenase that is most active in oxidizing ethanol has a very low Km for ethanol, approximately 0.04 mM, and is at more than 99% of its Vmax at the legal limit of blood alcohol concentration for driving (80 mg/ dL or about 17 mM). In contrast, the microsomal ethanol oxidizing system (MEOS) isozyme that is most active toward ethanol has a Km of approximately 11 mM. Thus, MEOS makes a greater contribution to ethanol oxidation and clearance from the blood at higher ethanol levels than at lower ones. Liver damage such as cirrhosis results partly from toxic by-products of ethanol oxidation generated by MEOS. Al Martini, who has a blood alcohol level of 240 mg/ dL (approximately 52 mM), is drinking enough to potentially cause liver damage, as well as his car accident and arrest for driving under the influence of alcohol. The various isozymes and polymorphisms of alcohol dehydrogenase and MEOS are discussed in more detail in Chapter 25.

how does glucagon signal transduction proceed?

The pathway for signal transduction by glucagon is one that is common to several hormones; the glucagon receptor is coupled to adenylate cyclase and cAMP production. Glucagon, through G-proteins, activates the membrane-bound adenylate cyclase, increasing the synthesis of the intracellular second messenger cAMP. cAMP activates PKA (cAMP-dependent protein kinase), which changes the activity of enzymes by phosphorylating them at specific serine residues. Phosphorylation activates some enzymes and inhibits others. The G-proteins, which couple the glucagon receptor to adenylate cyclase, are proteins in the plasma membrane that bind GTP and have dissociable subunits that interact with both the receptor and adenylate cyclase. In the absence of glucagon, the stimulatory Gs-protein complex binds GDP but cannot bind to the unoccupied receptor or adenylate cyclase . Once glucagon binds to the receptor, the receptor also binds the Gs-complex, which then releases GDP and binds GTP. The α-subunit then dissociates from the βy-subunits and binds to adenylate cyclase, thereby activating it. As the GTP on the α-subunit is hydrolyzed to GDP, the subunit dissociates and recomplexes with the β- and y-subunits. Only continued occupancy of the glucagon receptor can keep adenylate cyclase active.

Treatment of mature insulin (see Fig. 6.12) with denaturing agents, followed by renaturation, does not restore mature insulin activity (a result distinct from which occurred when the experiment was done with ribonuclease). Why does rena-turation of denatured mature insulin not result in restoration of biological activity?

Treatment of mature insulin (see Fig. 6.12) with denaturing agents, followed by renaturation, does not restore mature insulin activity (a result distinct from which occurred when the experiment was done with ribonuclease). Why does rena-turation of denatured mature insulin not result in restoration of biological activity?

how does proteolytic cleavage different than other forms of regulation?

it is irreversible

what is the Cori cycle?

pathway for lactate to regenerate glucose via a gluconeogenic pathway

what is the rate limiting enzyme in glycolysis?

phosphofructokinase-1

is glycogen phosphorylase active when phosphorylated or unphosphorylated?

phosphorylated by a kinase ↔ kinase is affected by endocrine regulation, adenylate cyclase, cAMP

what is the consequence of a free monosaccharide being phosphorylated at their terminal carbons?

prevents transport out of the cell (e.g. glucose 6-phosphate)

what are enzymes?

proteins that catalyze biochemical reactions by increasing the speed at which they occur by reducing the activation energy

what drives epi production at the same level as an MI?

severe hypoglycemia from basal of 34 → 1500 pg/ml

which types of hormones enter a cell to exert their effects?

steroid hormones

what is feedback regulation?

the end product of a pathway directly or indirectly controls its own rate of synthesis

what is signal transduction?

the mechanism by which the message carried by the hormone ultimately affects the rate of the regulatory enzyme in the target cell

what is the role of vitamins E and C?

they are oxidation-reduction coenzymes that act as antioxidants and protect against oxygen free radical injury

is glycogen synthase active when phosphorylated or unphosphorylated?

unphosphorylated by a phosphatase

The ketone bodies synthesized in the liver are β-hydroxybutyrate and acetoacetate. A third ketone body, acetone, is formed by the nonenzymatic decarboxylation of acetoacetate. Acetone is volatile and accounts for the sweet mousy odor in the breath of patients such as Di Abietes when they have a ketoacidosis. What functional groups are present in each of these ketone bodies?

β-Hydroxybutyrate and acetoacet ate are carboxylates (dissociated carboxylic acids). Aceto acetate and acetone contain keto/ ketone groups. Because β-hydroxy butyrate contains an alcohol (hydroxyl) group and not a keto group, the general name of ketone bodies for these compounds is really a misnomer.

what are some of the regulatory pathways of phosphorylation by cAMP dependent protein kinase?

↑active transport (in or out) ↑opening or closing of channesl ↑protein synthesis ↑DNA/RNA synthesis ↑glycogen synthesis ↑glycogen breakdown ↑lipid breakdown ↑microtubule secretion

glucagon and epi receptors are linked to Gs protein, what are the first intracellular products?

↑cAMP and ↑PKA active form

where does epinephrine have a impact glycolysis?

↑epi → ↑protein kinase A → ↓rate of rxn of pyruvate kinase (PEP → pyruvate)

what metabolic actions ∆ when epi binds to its receptor?

↑glycogenolysis ↑lipolysis and ketosis ↓glucose utilization ↑insulin secretion ↑glucagon secretion *counterintuitive?