BIOCH 100: Enzymes

What is a Lineweaver-Burk Plot?

-Linear transformation of MM euqation and plot (aka double reciprocal plot) -when Vo is plotted against [S], it's not always possible to determine when Vmax has been achieved b/c of gradual upward slope of hyperbolic curve at high substrate concentrations. However, if 1/Vo is plotted vs. 1/[S], straight line obtained. Good for caluclation of Km and Vmax as well as to determine mechanism of action of enzyme inhibitors

What can many disease that cause _____ do? What can this tell you?

-Many diseases that cause tissue damage result in ^ release of intracellular enzymes into plasma. -activities of many of these enzymes routinely determined for diagnostic purposes -level of specific enzyme activity frequently correlates w/ extent of tissue damage.--> t/f determining degree of elevation of particular eznyme activity in plasma often useful in evaluating patient prognosis

What 3 conclusions can we make about the MM equation?

1. Km charactersitics=Km, the michalis constant, is charactersitic of an eznyme and its particular substrate and reflects the affinity of the enzyme for the substrate. Km=[S] @ which reaction velocity is equal to one half Vmax (Km=[S] at which initial rate (V0)=1/2Vmax (Km is independent of [E]!!, does not vary w/ enzyme conc.) IMPORTANT: a. The lower the Km, the higher the affinity of E for S (b/c low concentration of substrate needed to half saturate (to reach 1/2Vmax) b. The higher the Km, the weaker the affinity (hexokinase achieves 1/2 Vmax at lower [S]=glucose than glucokinase w/ higher Km) high conc of substrate needed to half-saturate 2. Reaction rate directly proprotional to [E] b/c [S] is not limiting. Ex: if [E] is halved, Vo (for given [S]) and Vmax are halved 3. Reaction order: When [S] <<< Km, the veloicty of the reaction is approx proportional to [S] = first order reaction w/ respect to substrate. When [S] >>> Km, reaction rate=Vmax=zero order reaction (reaction rate independent of [S]-- enzyme is saturated w/ substrate)

What factors are responsible for catalytic efficiency of enzymes (3)?

1. TS stabilization 2. catalysis 3. TS visualization

What must we assume w/ MM?

1. [S] is much greater than [E] so that perecentage of total substrate bound by enzyme at any one time is small ( [E]<<<[S]) 2. Reaction is at STEADY STATE. Concentration of ES complex does not change w/ time, that is, rate of formation of ES (E+S->ES) is equal to that of the breakdown of ES (to E + S and to E + P). In general, an intermediate in a series of reaction sis said to be in steady state when its rate of synthesis is equal to its rate of degradation (rate of synthesis of ES=rate of degradation of ES) 3. Initial reaction velocities (Vo) are used in anaylsis of enzyme reactions. This means that rate of reaction is measured as soon as enzyme and substrate are mixed. At that time, concentration of product is very small, and therefore, the rate of the back reaction from product to substrate can be ignored (k-2 is VERY SMALL)

How are enzymes named systematically? *** KNOW THE 6 CLASSES ABOVE!

1. enzymes divided into six major classes, each w/ numerous subgroups 2. for given eznyme, suffix -ase is attached to fairly complete description of chemical reaction catalyzed including names of substrates

What are two ways in which you can understand the mechanism of action of enzymes?

1. enzymes provide alternate energetically favorable reaction pathway dif from uncatalyzed reaction. 2. how active site chemically facilitates cataylsis

What is the optimum temp range for most human enzymes? In bacteria?

1. humans=35C-40C 2. thermophilic bacteria in hot springs=70C

Substrate concentration (maximal velocity/velocity, shape of enzyme kinetics curve)

1. maximal velocity=rate or veloicty of reaction (v) is number of substrate molecules converted to product per unit time. Veloicty usually expressed umol of product formed per min. Rate of enzyme-cat reaction increases w/ substrate concentration until a maximal velocity (Vmax) reached. Leveling off of reactionr ate at high substrate concentration reflects saturation w/ substrate of all available binding sites on enzyme molecules present 2. Most eznymes show Michaelis-Mentin kinetics, in which plot of initial reaction velocity (Vo) against substrate concentration is hyperbolic. In contrast allosteric enzymes do not follow MM kinetics and show sigmoidal curve

What are the nonprotein componnets needed for enzymes?

1. metal ions (eg Ni++) 2. coenzymes- organic molecules (TPP) 3. cosubstrate- organic and charged (NAD+) 4. prosthetic groups- very tightly (usually covalently) attached to protein (eg pyridoxal phosphate, biotin)

Competitive inhibition (What is it? effect on Vmax, effect on Km, effect on LBP)

1. occurs when inhibitor binds reversible to same site that substrate would normally occupy and therefore competes w/ substrate for that site 2. effect of comp inhib is reversed by increasing [S]. At sufficiently high [S], the reaction velocity reaches Vmax observed in absence of inhibitor, that is, Vmax is unchanged 3. Comp inhib increases apparent Km for a given substrate. Means that, in presence of comp inhib, more substrate is needed to achieve on half Vmax 4. plots of inhibited and unhibited reactions intersect on y axis at 1/Vmax (Vmax unchanged). The inhibited and unhibited reactions show dif x-axis intercepts, indacting that apparent Km is increased in presecen of competitive inhibitor b/c -1/Km moves closer to zero from a negative value

What are the 6 types of enzymes? define them

1. oxidoreductase-catalyzes oxidation-reduction reaction 2. transferase- catalyzes transfer of C-, N-, P-containing groups 3. hydrolase- catalyzes cleavage of bonds by addition of water 4. lyase- catalyzes cleavage of C-C, C-S, and certain C-N bonds 5. isomerase- catalyzes racemization of optical or geometric isomers 6. ligase- catalyzes formation of bonds carbon and O, S, N, coupled to hydrolysis of high energy phosphate

pH (3 things)

1. pH effect on active site ionization=concentraiton of protons (H+) affects reaction velocity in several ways: a. catalytic process usually requires that enzyme and substrate have specific chem groups in either ionized or unionized state in order to interact. (i.e. catalytic activity may require NH3+ to be protonated, but at alkaline pH it's not, so rate of reaction declines) 2. pH effect on enzyme denaturation=extreme pH can lead to denaturation b/c structure catallytically active protein molecule depends on ionic character of AA side chains 3. variable pH optimum= pH at which maximal enzyme activity achieved is dif for dif enzymes, often reflects [H+] at which enzyme functions in body. (i.e. pepsin is maximally active at pH 2 in stomach) (combo of 1 and 2)

Noncompetitive inhibition (What is it? effect on Vmax, effect on Km, effect on LBP)

1. recognized by charactersitic effect on Vmax. occurs when the inhibitor and substrate bind at dif sites on enzyme. Noncompetitive inhibitor can bind either free eznyme or enzyme-substrate complex, thereby preventing reaction from occuring 2. noncomp cannot be overcome by increasing [S]. Therefore, noncomp inhibitors decrease the apparent Vmax of the reaction 3. do not interfere w/ binding of substrate to enzyme. Therefore, enzyme shows same Km in presence or absence of noncomp inhibitor (Km=unchanged) 4. Apparent vmax decreases in presence of noncomp inhibitor whereas Km unchanged

What are allosteric enzymes regulated by? How are they usually composed? Positive vs. negative effectors? What do they frequently catalyze?

1. regulated by molecules called effectors that bind noncovlaently at a site other than active site. 2. almost always composed of multiple subunits and regulatory (allosteric) site that binds the effector is distinct from the substrate-binding site and may be located on a subunit that is not itself catalytic 3. effectors that inhibit enzyme activity=negative effectors, whereas those that increase enzyme activity are called positive effectors. -- they can affect the affinity of the enzyme for its substrate (K_.05, modify the maximal catalytic activity of the enzmye (Vmax), or both 4. freq. catalyze the committed step, often the rate-limited step, early in a pathway

Two ways enzymes are named

1. short, convienent 2. systematic name

What factors can affect reaction velocity?

1. substrate concentration 2. temperature 3. pH

Temperature (increase/decrease)

1. velocity increase w/ temperature=reaction velocity increases w/ temp until peak velocity reached. This increase is result of increased number of substrate moelcuels having sufficient energy to pass over energy barrier and form products of reaction 2. velocity decrease w/ higher temperature=futher elevation casues decrease in reaction veloicty as result of temperature-induced denaturation of enzyme *bell shaped curve

Catalytic efficiency

10³ - 10⁸ times faster reactions than without enzyme turnover number of Kcat=the number of substrate molecules converted to per second (know this)

Jon Jakob Berzelius

1825- discovered catalytic effect of enzymes

James Sumner

1926- isolated first enzyme in pure form

Northrup, Stanley, Sumner

1947- awarded the Nobel prize for isolation of enzyme pepsin

Linus Pauling

1948- enzymes are molecules that are complementary in structure to activated complexes of reactions they catalyze

How many drugs of the most common are...?

5 of 10 most common prescribed drugs in US are enzyme inhibitors. ex: Beta-lactam antibitoics (penicillin and amoxicillin) act by inhibiting enzymes involved in bacterial cell wall synthesis.

What is the equation for an enzyme? Draw graph on page 55 in textbook. where does deltaG occur

A<->T*<->B change in energy **NOTE THAT REACTIONS ARE REVERSIBLE!

Catalysis

AS can provide catalytic groups that enhance probability that TS is formed. In some enzymes, these groups can participate in general acid-base catalysis in which AA residues provide or accept protons. In other enzymes, catalysis may involve transient formation of covalent ES complex

What is an example of an allosteric enzyme? What is it involved in?

Aspartate transcarbamoylase (regulated enzyme in pyrimidine biosynth) ATP (R sites) and/or substrate (C sites)-> "R" state CTP or UTP (R sites)-> "T" state (**like chargaff's principle?? listent to lecture)

What is the MM equation

Describes how reaction velocity varies w/ substrate concentration

How does an enzyme allow the reaciton rate to be lowered? Does it change equilibrium?

Enzyme allows a reaction to proceed rapidly under conditions prevailing in cell by providing an alternate reaction pathway with a lower Ea. Enzyme does not change the free energies of the reactants (substrates) or products, and therefore DOES NOT CHANGE THE EQUILIBRIUM OF THE REACTION! It does, however, accelerate the rate by which equilibrium is reached

Describe the rate of reaction and what defines it

For molecules to react, must contain suffiecient energy to overcome the energy barrier of transition state. IN absence of enzyme, only small proportion of a population of moelcuels may posses enough energy to acheive the T*. Rate is dtermined by number of such energized moelcules. In general, the lower the Ea, the more molecules have suffieicient energy to pass thru the transition state and theorfore, the faster the rate of reaction

Catalytic groups in enzmye

Help T* formation. can be acid/base catalysis (donation and acceptance of protons) or by forming covalent enzyme-substrate complex can use some binding energy to alter conformation of substrate

What can drugs also act to do?

Inihibit extracellular reactions. Illustrated by angiotensin-converting enzyme (ACE) inhibitors. Lower BP by blocking plasma ACE that cleaves angiotensin I to form potent vasoconstrictor angiotensin II. Includes captopril, enalapril, and lisinopril (cause vasodilation->reduce blood pressure); aspirin irreversibly inhibits prostaglandin and thromboxane synthesis by inhibiting cycloooxygenase. Other non-steroidal anti-inflammatory drugs (NSAIDS) are reversible inhibitors of same enzymes (ibuprofen).

Are enzymes all over or in a specific spot? Give examples (4)

Many enzymes are localized in specific organelles within the cell. this compartimentalization serves to isolate the reaction substrate or product from other comepting reactions. This provides favorable environment for the reaction and organizes the thousands of enzymes present in the cell into purposeful pathways 1. mitochondria=TCA cycle, fatty acid oxidation, oxidation of pyruvate 2. cytosol=glycolysis, PP pathway (pentose phosphate), fatty acid synthesis 3. nucleus=DNA and RNA synthesis

What is an example of competitive inhibitors clinically? Describe how they work!

Statin drugs-- this group of antihperlipidemic agents complettively inhibits the rate-limiting (slowest) step in cholesterol biosynthesis. This reaction is catalyzed by hydroxymethylglutaryl coenzyme A reductase (HMG CoA reductase). Statins, such as atorvastatin (lipitor) and pravastatin (pravachol) are structural analogs of the natural substrate for this enzyme and compete effectively to inhibit HMG CoA reductase. By doing so, they inhibit de novo cholesterol synthesis, thereby lowering plasma cholesterol levels

Why is there a small dip in the curve at ES?

Substrate bound to enzyme (ES) is at slightly lower energy than unbound substrate (S) and explain small dip in curve at ES

How can plasma enzymes be classified? how are enzyme levels in health individuals

Two major groups: 1. relatively small group of enzymes are actively secreted into blood by certain cell types (i.e. liver secretes zymogens of enzymes involved in blood coag 2. large number of enzymes species are released from cells during normal cell turnover (these enzymes almost always function intracellularly and have no use in plasma) in healthy individuals: levels of enzymes are fairly constant and represent steady state in which rate of release from damaged cells into bplasma is balanced by equal rate of removal from plasma. Increased plasma levels of these may indicated itssue damage (cell necrosis as result rof disease or trauma)

TS stabilization

active site often acts as flexible molecular template that binds to substrate and initates its conversion to TS, a structure in which the bonds are not like those in the substrate or product. By stabilizing the TS, the enzyme greatly increases concentration of reactive intermediate that can be converted to product and thus accelerate reaction (TS CANNOT BE ISOLATED)

Describe mechanism of action of chymotrypsin

an eznyme of portein digestion in intesting, includes general base, general acid, and covalent catalysis. A histidine at active site of enzyme gains (general base) and loses (general acid) protons, mediated by pK of histidine in proteins being close to phsiologic pH. Serine at active site forms transient covalent bond w/ substrate

inhibitor

any substance that can decrease the velocity of an enzyme-catalyzed reaction; reversible or irreversible (bind thru covalent bonds)

Active site

cleft or pocket lined with amino acids that form a 3D surface that is shaped complimentary to substrate, amino acid side chains in active site bind to regions of substrate; binding causes conformation change=induced fit model

cosubstrate

coenzymes that only transiently associate w/ enzyme; dissociate from eznyme in altered state (i.e. NAD+)

How do differences in isozymes (say what kind) affect testing? GIve example

differences in amino acids may cause change in charge of enzyme, allowing electrophoresis to separate them. Ex: plasma levels of creatine kinase (CK) are commonly determined in diagnosis of myocardial infarction (MI). They are mparticularly useful when ECG is difficult to interpret such as when there have bene previous episodes of heart disease

What do virtually all chemical reactions have?

energgy barrier separating the reactants and products called Ea (activation energy)=energy dif btwn that of reactants and a high energy intermediate (transition state T*), which is formed during conversion

Holoenzyme

enzyme protein plus its non-protein component

apoenzyme

enzyme protein without its non-protein component

What does the MM model require of the enzyme reaction? Show the reaction equation involving 1 substrate

enzyme reversibly combines w/ substrate and then regenerates free enzyme

TS visualization (analogy)

enzyme-catalyzed conversion of substrate to product can be visualized as being similar to removing a sweater from uncooperative infant. Process has high Ea because the only reasonable strategy for removing garment requires random flailing of baby results in both arms being fully extended over head, an unlikely posture. However, we can envision a parent acting as an enzyme (ES), first coming in contact w/ baby and then guiding baby's arms into extend, vertical position, analogous to TS.

Stabilization of transition state

enzymes binds substrate in a structure that resembles the transition state. this lowers the free energy of activation, puts more molecules into the T* form and accelerates the reaction rate

What are isozymes?

enzymes that catalyze same reaction but have dif physical properties. Dif organs have dif proportions of dif isozymes

~Book/Main lecture~ Enzymes

enzymes=protein catalysts that increase the rate of reactions w/o being changed in overall process

Why is regulation of reaction velocity of enzymes essential? The rate of most enzymes are responsive to....

essential if organism is to coordinate its numerous metabolic processes ...changes in [S] b/c intracellular level of many substrates is in range of Km. Thus, an increase in [S] prompts an increase in reaction rate, which tends to return [S] toward normal. In addition, some enzymes w/ specialized regulatory functions respond to allosteric effectors and/or covalent modification or they show atlered rates of enzyme synthesis (or degrdation) when physiological conditions change

What does lead do to heme synthesis ?

forms covalent bonds w/ sulfhydryl side chain of cysteine in proteins. Ferrochelatase, enzyme involved in heme synthesis is irreversibly inhibited by lead

prostethic group

if coenzyme is permanently associated w/ eznyme and returned to its original form; very tight (usually covalent bond)

Some enzymes show relatively high activity..... Give example! How is this important clinically?

in only one or a few tissues. T/f presence of increased levels of these enzymes in plasma refelcts damage to corresponding tissue. Ex: alanine aminotransferase is abundant in liver. Elevated ALT in plasma signals possible damage to hepatic tissue. Increase in plasma levels of enzymes w/ wide tissue dsitribution prokvide less specific indication of site of cell injury

Can enzyme activity be regulated? How?

increased or decreased, so that the rate of product formation responds to cellular need

What are commonly used statins?

lovastatin (Mevacor, Altocor), atorvastatin (lipitor), and simvastatin (zocor). Figure in previous card shows pravastatin (Pravachol)

Covalent modification (general description, describe phosphory/dephosphor, how do enzymes respond?)

many enzymes regulated by this, most often by addition or removeal of phosphate groups from specific serine, threonine, or tyrosine residues of eznyme. Protein phosphorylation is recongized as one of primary ways in which cellular processes are regulated 1. phosphorylation and dephosphorylation (usually constitutive): phoshphorylation reactions catalyzed by family of enymes called protein kinases that use ATP as the phosphate donor. Phosphate groups are cleaved from phosphorylated enzymes by action of phosphoprotein phosphatases 2. enzyme response to phosphorylation: depending on specific enzyme, the phosphorylated form may be more or less active than the unphosphorylated enzyme. Ex: hormone-mediated phosphorylation of glcyogen phosphorylase (enzyme that degrades glycogen) increases activity, whereas phosphor of glycogen synthase (eznyme that syntehsizes glyocgen) decreases activity

Isozymes and quaternary structure (example! Describe dif forms and where they are found)

many isoenzymes contain dif subunits in various combos ex; CK occurs as three isoenzymes. Each is dimer composed of two polypeptides (B and M subunits) in one of the combos: CK1= BB, CK2=MB CK3=MM. Each CK shows dif electrophoretic mobility (virtually al CK in brain is BB, whereas MM=skeletal muscle, In cardiac muscle 1/3=MB, rest is MM

What can be used for diagnosing MI? how?

measurement of blood levels of proteins w/ cardiac specifictiy -myocardial muscle is the only tissue that contains >5% of total CK activity as the CK2 (MB) isoenzyme. Appearance of this hybrid isoenzyme in plasma is virutally specific for infarction of myocardium. -Following an acute MI, CK2 appears in plasma w/in 4-8 hours following onset of chest pain, reaches peak of activity at ~24 hours, and returns to baes line after 48-72. -Troponins T (Tnt) and I (TnI) are regulatory proteins involved in muscle contractility. Cardiac-speicifc isoforms (cTn) are released into plasma in repsonse to cardiac damage. They are highly sensitive and psecific for damage to cardiac tissue. cTn appear in plasma w/in 4-6 hours after an MI, peak in 24-26 hours, and remain elevated for 3-10 days. *ELEVATED cTn, in cmbo w/ clinical presentation and charactersitic changes in ECG, are considered gold standadr in mI diagnosis)

What is an important group of irreversible inhibitors?

mechanism-based inhibitors that are converted by enzyme itself to a form that covalently links to enzyme, thereby inhibiting it (suicide inhibitors)

What is oxypurinol?

metabolite of the prodrug allopurinol, it's a noncomp inhibitor of xanthine oxidase, an eznyme of purine degradation; used to treat GOUT

What is lead considered?

noncomeptitive inhibitor of ferrochelatase

What do some enzymes require?

nonproteins for enzymic activity

Describe the active site

not passive receptacle for binding substrate, but a complex molecular machine employing a diversity of chemical mechanisms to facilitate conversion

What are the optimal pHs for pepsin, trypsin and alkaline phosphatase?

pepsin=2, trypsin=~5.5-6, AP=~8.5

What is plasma? What can it be used for? Plasma v. serum

plasma=fluid noncellular part of blood. Lab assays of enzyme activity often use serum (which is obtained by centriguation of whole blood after it has been allowed to coagulate). Plasma is phsyiologic fludi whereas serum is prepared in lab

How can enzymes be regulated?

positively or negatively

Enzyme

protein (or rarely RNA=ribozymes) catalyst that increases velocity of reaciton w/o being consumed in reaction

How can cells also regulate enzyme activity? Describe dec/inc of activity

regulating the amount of enzyme present by: 1. rate of enzyme degradation 2. (more typically)- the rate of enzyme synthesis *increase (induction) or decrease (repression of enzyme synthesis leads to an alteration in the total population of active sites

Subcellular localization

separates reaction pathways, allows macromolecular enzyme complexes to for that favor overall pathways

Specificity

single molecule or group of structurally related substrate molecules

Lineweaver-Burk plot and equation

slope=Km/Vmax

What can some RNA do?

some RNA can catalyze reactions that affect phosphodiester and peptide bonds= ribozymes

How are enzymes shortly named?

suffix -ase attached to substrate of reaction or to description of action performed (lactate dehydrogenase)-- some tho retain original tirival names like trypsin and pepsin which doesn't give any hints

Synthetase vs synthase and phosphatase vs phosphorylase? Also... dehydrogenase, oxidase, and oxygenase

synthetase=requires ATP, synthase=no ATP required, phosphatase=uses water to remove phosphate group, phosphorylase=uses inorganic phosphate to break a bond and generate a phosphorylated product dehydrogenase=NAD+ or falvin adenine dinucleotide [FAD] is an electron acceptor in a redox reaction oxidase=oxygen is the acceptor, and oxygen atoms are not incoporated into substrate oxygenase=one or both oxygen atoms are incorporated

Activation energy (why are uncatalyzed reactions often slow? another name? This is true even though...)

the peak of energy is the difference in free energy btwn the reactant and T*, in which high-energy, short-lived intermediate is formed during conversion of react to produce. Because of the high Ea, the rates of uncat chemical reactions are often slow another name=free energy of activation (deltaG^cross)

Enzymes subject to regulation of synthesis are often.... (example) In contrast.....enzymes that are in constant use are usually.... How do the rates of these actions compare?

those needed at only one stage of devleopment or under selected physiologic conditions. Ex: elevated levels of insulin as result of high blood glucose levels cause increase in synth of key enzymes invovled in glucose metabolism. ....not regulated by altering the rate of enzyme synthesis. Alteration in eznyme levels as a result of induction or repression of protein synth are slow (hours to days) compared w/ allosterically or covalently regulated changes in enzyme activity which occur in seconds to minutes

How do reversible inhibitors bind? What are the most common types?

through noncovalent bonds and thus dilution of the enzyme-inhibitor complex results in dissociation of the inhibitor and recover of enzyme activity. Most common are competitive and noncompeitive

What is an important group of competitive inhibitors?

transition state analogs, stable molecules that approximate the structure of the TS and therofore bind the enzyme more tightly than the substrate

What are coenzymes commonly dervied from? Give two examples.

vitamins ex: 1. NAD+ contains niacin 2. FAD contains riboflavin

What are heterotropic effectors? What do they lead to? Are they common? give an example.

when effector is dif from substrate. Example: feedback inhibitor-> enzyme converts D to E has allosteric site that binds end product G. If concentration of G increases (for example, b/c not used as rapidly as it is synthesized), the first irreversible step unique to pathway is typically inhibited. -feedback inhibition provides the cell w/ appropriate amounts of a product it needs by regulating the flow of substrate molecules thru the pathway that synthesizes the product. -Commonly encountered -Ex: glycolytic enzyme phosphofructokinase-1 is allosterically inhibited by citrate, which is not a substrate for the enzyme

Cofactor

when nonprotein moeity (for holoenzyme)=metal ion

coenzyme

when nonprotein moeity=organic molecule s

What are homotropic effectors? WHat do they cause? What do the binding sites exhibit?

when substrate itself serves as effector. -most often, allosteric substrate functions as positive effector. In such a case, presence of substrate molecule at one site on enzyme enhances the catalytic properties of the other substrate-binding sites. That is, their binding sites exhibit cooperativity.