Exam 1

Quaternary Structure: multi--subunit proteins: - Many proteins are made up of one or more subunits that self-associate into oligomers and polymers - Proteins are made up of L-amino acids and are thus _________ _________ (ie no points or mirror planes of symmetry) - subunits are most often arranged by rotations and ________________ (eg actin) - self-association also conserves _____ in extremely crowded cells

intrinsically asymmetric; translations; water

Substrate Binds to Enzyme's Active Site: - Active Sites are shallow depressions, into which substrates bind. - With a volume of ~1 nm3 , active sites are big enough to accommodate molecules that are ~1000 unified atomic mass units - Limited size puts the binding energy in a "Goldilocks range": Not too strong, Not too weak - just right. - the more energy released, the ____ stable state - Such sites are frequently stereospecific. - Chymotrypsin: a digestive enzyme that _______ proteins within your small intestine

stereospecific; more: cleaves

Structural Organization of Collagen Fibril: - Tropocollagen forms collagen fibers - Accounts for _______ observed in electron micrographs of uranium-stained collagen. - Head-to-Tail Self-Assembly, with individual tropocollagen trimers shown as arrows - Collagen fiber is further strengthened by: - ___________ interactions between neighboring tropocollagen units by ________ involving Hydroxy-Pro. - It is also covalently cross-linked (lysyl residues are oxidized and link to other lysyl residues)

striations; Noncovalent; H-bonds

Edman Sequencing: Repetitive removal of AA from ___________ & subsequent identification: • R is different for each amino acid • PTH-AA is identified by gas or liquid chromatography • Process is repeated over and over for 40-50 cycles • Automated sequencing of 5-20 µg peptide/protein • Revolutionized biochemistry - end up generating a polypeptide that is one amino acid ________

N-terminus; shorter

b-Sheet: Structural Features: - Pauling and Corey (first observed in b-keratins): - Polypeptide zig-zags to form an extended ______ referred to as a b-sheet. - R-groups point into and out of the plane of the sheet. - Typical torsion angles: phi = -140 omega = 130

conformation

Derivation of Michaelis-Menten Equation: Assumptions 1. [S]tot >> [E]tot , such that: [S]tot = __________ ≈ [S]free 2. Conservation of enzyme, such that: [E]tot = _______ 3. [P] = 0 throughout, measurements such that: "EX-to-E+P" is effectively ___________ 4. Enzyme remains fully _______ throughout measurements.

([S]free + [EX]); [E] + [EX]; unidirectional; active

Role of H-Bonding in a- & b-Structures: - Hydrogen-bonding per se is NOT a major driving force in protein folding. - Why? 2 H-bonds are broken. 2 H-bonds are formed so overall energy will be ____ - HOWEVER, formation of extended H-bond networks act as structure-___________ elements. - a-Helix or b-Sheet creates scaffold that helps _________ protein's interior by positioning side-chains to maximize hydrophobic interactions (hence maximize H2O release). - Intra- and inter-chain H-bonding compensates for loss of R-N···H···OH2 & HOH···O=C-R H-bonds.

0; organizing; dehydrates;

- liquid water has ______ h bonds on average whereas ice has _____ h bonds - water has a high heat capacity due to its _________ ______ which is resistant to changes in temperature - water has a high BP relative to its ________ _______ - water has a high ________ relative to Its MW - water has _______ interactions with ionic molecules - water has special interactions with nonpolar molecules

3.4; 4; thermal mass; molecular weight; viscosity; strong

Sugars adopt cyclic structures: - in aqueous solution, aldotetraoses and all monosaccharides with ___ or more carbons are predominantly ______ - for hexoses, OH group at ____ reacts with the carbonyl to form either a hemiacetal or a hemiketal, creating an additional ______ ______

5; cyclic; C5; chiral center

Noncovalent interactions are weaker than DG of 144 -360 kJ/mol for bond energies of C-C single bond: - hydrogen bonds are basically an ________ - ____________ interactions: attractive and repulsive forces - salt bridge are H bonding + _____________ - Hydrophobic interactions are H2O release from ________ groups, driven by increase in DS - Pi stacking interactions is Stacking of aromatic rings (__________ pi electrons) - van der Waals interactions are Weak bonding interaction between oscillating dipoles

A/B rxn; electrostatic; electrostatics; apolar; delocalizes

Enzymes are grouped into six classes: 1. Oxidoreductases - catalyze oxidation-reduction reactions. Ethanol + NAD+ ⇌ ___________ + NADH + H+ 2. Transferases - catalyze transfer of __________ groups. Glucose + ATP ⇌ Glucose-6-P + ADP 3. Hydrolases - catalyze hydrolysis of ________ bonds. Glucose-6-P + H2O ⇌ Glucose + Pi 4. Lyases - catalyze elimination/addition of a functional group. Malate ⇌ H2O + __________ 5. Isomerases - catalyze ___________ rearrangements. Glucose-6-P(ald) ⇌ Fructose-6-P(keto) 6. Ligases - catalyze __________ of molecules Glutamate + NH3 + ATP ⇌ _________ + ADP + Pi 7. Ergases - convert chemical bond energy into __________ work (F✕d). ATP + Myosin at Position-1 ⇌ Myosin at Position-2 + ADP + Pi

Acetaldehyde; functional; covalent; Fumarate; intramolecular; joining; Glutamine; mechanical

Enzymes catalyze chemical reactions by exploiting the properties of: - Acidic residues - ____ _____ & C-terminal carboxyl groups - Basic Residues- _____ _____ & N-terminal amino groups - Nucleophilic residues - Lys, His, _____ ____ - Metal ions - Lewis _____ that alter substrate reactivity - Coenzymes - organic molecules (often derived from vitamins) that greatly expand catalytic ________ - Arginine's basic side-chain - positive charge over entire physiologic pH range; helps bind __________ substrates) - Even hydrophobic groups - help by binding ______ substrates and altering active-site acid/base behavior. As a catalyst, an enzyme always returns to its original form after each round of catalysis.

Asp, Glu; Lys, His; Cys, "Ser"; acids; versatility; anionic; nonpolar

Fully active proteins must fold on a biologically relevant timescale: - ___________, for example, can double in number in as little as 18 minutes, with about 2500 different proteins produced throughout a microorganism's growth cycle. - Proves that proteins must fold within minutes, or less. - Most small proteins fold _____________ into a functionally active (fully folded) conformation. (No assistance needed) - It's estimated that around 5% of all polypeptides emerging from ribosomes will fail to fold and/or require ___________. - Indeed, many proteins need help to fold, so much so that cells produce a set of "________" (i.e., ATP-dependent mechanoenzymes that facilitate protein folding). - Called molecular __________, these proteins help their "client" proteins to refold into their active conformation.

Bacteria; spontaneously; assistance; foldases; chaperones

Torsion angles of Peptide Bond: - The three main torsion angles of polypeptide backbone: phi _______ psi _________ (0 or 180) omega _________ (peptide bond) • trans-________ of peptide bond restricts omega to 180° in nearly all the main-chain peptide bonds. • In rare cases, omega = 0° for a cis peptide bond, which almost always involves ________

C-N; O=c-c(alpha); O=C-N; planarity; proline

Prion Diseases: - Found in _____ of infected individuals. - Function of normal prion protein (PrPC) remains unknown. - Aberrant prion (PrPSc) has an infectious _______. - PrPSc promotes PrPC misfolding: - Misfolded prion protein then aggregates into _______ fibrils that destroy neurons and our capacity to learn.

CNS; b-fold; amyloid

- Hb & Mb are both 8-helix polypeptides. - Heme group lodged between Helices _______. - Heme groups in Hb don't touch each other, and O2 binding behavior depends on ________ interactions

E and F; subunit

Secondary structure: - Defined as helix and sheet structures that arise from the formation of regularly spaced ___________between main-chain atoms. - Principal secondary structures include: • a-Helix (also form Proline Helix, 3,10-Helix, and Collagen Triple Helix) • Parallel & Antiparallel b-Pleated Sheets • Turns - short sequences that connect helices and/or sheets & allow proteins to be _______ and stable.

H-bonding; compact

Meaning of Michaelis Constant, Km: - Km is a kinetic parameter that may or may not equal ___, the latter being a _______________ parameter. Only Kd is a measure of affinity. (_____ Kd means High Affinity) - Km = Kd only when k2 >> k3 (i.e., when S _______/ release is ______) - Km = - Kd =

Kd; thermodynamic; low; binding; rapid; (k2 + k3)/k1; k2/k1

- _________= defined as any molecule that binds to binding sites on receptors, transport proteins, or enzymes. - Examples: Enzymes: Substrates, Inhibitors & Activators Receptors: Agonists & Antagonists - High Kd = _______ binding; Low Kd = strong binding - Extent of binding is ______, whenever [ L] > Kd - Extent of binding is low, whenever [L] < Kd

Ligand; weak; high

AA's with Positively Charged Side-chains: - Basic Amino Acids: ___________ have significant side-chain positive charge at pH 7.0. - ___________ is the only basic AA with an ionizable group near neutrality: can serve both as H+-donor or H+- acceptor in enzyme-catalyzed reactions.

Lys, Arg and His; histidine

What do we mean by chemical reaction rate? - chemical reaction rate is always expressed as ______ - Therefore rate constants must have units that allow reaction rate to have the above units

M/sec;

Folding Funnel Depicts Protein Folding: - Folding is a massive parallel search of conformation space, proceeding thru multiple paths: ALL PATHS PROCEED IN ________, CONVERGING AS FOLDED STUCTURES GAIN GREATER STABILITY. - Pauses represent refolding intermediates. - entropy ________ but overall is increasing bc H20 release and hydrophobic interactions are _________

PARALLEL; decreases; dominant

It all begins with the Peptide Bond: • ________ & _______ analyzed geometry and dimensions of peptide bonds in crystal structures of simple molecules containing one or several peptide bonds. • They obtained consensus bond-________ and bond angles • C-N peptide bond length is consistently __________ than the C-N bond in most amides.

Pauling; Corey; lengths; shorter

H-Bonds are found throughout Nature: - ESSENTIAL FOR BIO-SPECIFICITY: - Enzyme-substrate interactions - ________________ interactions - ____________ base-pairing - H-Bonds are easy to make & just as easy to break (fast kinetics)

Receptor-ligand; RNA & DNA

Planarity of peptide bond is key to Life: • C-N bond has 40% double-bond character • _________ explains shorter bond-length 1.32 Å for peptide versus 1.53 Å for simple amides • C=O and N-H atoms within the peptide bond are approximately _________ • Allows proteins to fold on biologically feasible timescale. • Reduced freedom greatly facilitates protein __________. - Each peptide bond behaves as a ______ unit - Less rotational flexibility around _____ bond • The amino acid side-chains direct the folding of the nascent polypeptide into a functional protein and do much to stabilize that final conformation.

Resonance; coplanar; folding; planar; C-N

Tropocollagen has a Unique Triple Helix: - Collagen plays a central role in the mechanical strength of tissues. • _____-shaped molecule (3000 Å length ´ 15 Å diameter) • Structural building block is Tropocollagen • Mechanical strength arises from _________ between aligned molecules. - Characteristics: • Tripeptide pattern beginning with ____. • ___ frequently follows Gly. • Unusual amino-acid: Hydroxyproline (Hyp)

Rod; X-linking; Gly; Pro

AA sequence is determined in two ways: a. cDNA Sequencing (covered later) b. Enzymatic/Chemical Fragmentation, followed by Fragment ________ & Automated Sequencing: - the pure protein can a) use specific protease or b) use ________ ________ (react with ______ and treated with acid) to get to a specific set of peptide fragments - ________ cleaves on carboxyl side of _____ and _______ - Chymotrypsin-cleaves on carboxyl side of __________ a.a's - Amino Acid sequences are always written with N-terminus on the left, Cleavage occurs "AFTER" the specific residue, freeing its _______ group

Separation; cyanogen bromide; Met; trypsin; arg; lys; aromatic; COOH

Determining Km and Vm experimentally 1. Keep ________ constant during the entire experiment. 2. Use ___________ concentration [S1]. 3. Measure Product formed during the _______ (or initial-rate) phase. 4. Draw best straight-line through data points to estimate: = _______ 5. Repeat steps 2, 3, and 4 for each different substrate concentration. 6. Graph data points in plot of _______ versus [S].

[Etotal]; substrate; linear; v= delta [P]/delta t; velocity

- Aromatic groups can _________ UV light - (trp>tyr>phe) uv absorbance - ____ group of Tyr and _____ group of Trp can form H bonds to play catalytic roles in some enzymes - polar R groups form _________ with water

absorb; OH; NH; h bonds

Metal Ion Catalysis: - Depends on the ability of the metal ion to serve as a template, a lewis _____ or a redox ______ - to bind and orient _________ - _______________ negative charge in TS - Increases the ______ of Bound Water or Alcohols Notice that metal ion is a Lewis Acid (or electron pair acceptor) - Mediates ______ Reactions since the metal ion changes its oxidation state

acid; reactant; substrates; shield/stabilize; redox

Syncatalytic conformation changes: - - Enzyme and substrate(s) jointly reach and surmount the transition-state. - Conformational flexibility allows enzyme to ______ its shape, stabilizing each intermediate throughout the catalytic reaction cycle. - Notice how reactant molecules follow the lowest-lying energy path, maximizing _____ of product formation. - Shows that the enzyme is _______, not static as predicted by "Lock in-Key" mechanism.

adjust; rate; dynamic

Enzyme interacts preferentially with transition-state species: • Enzymes bind transition state with greater ______ than either substrates or products - Increases the rates of forward and backward steps _______ • Important corollary - good substrates shouldn't bind too tightly - Don't want to get stuck in an ES complex. • Transition-state ________ are good inhibitors of enzymes - As chemically stable mimics of the stereo-electronic properties of enzyme transition states, certain molecules can be remarkably effective inhibitors. (THE BASIS FOR RATIONAL _____ DESIGN)

affinity; equally; analogues; DRUG

Enzymes Catalyze Reactions: • Play pivotal roles in ____ biochemical processes • As true for all catalysts, they increase reactivity (reaction rate) without altering equilibrium (________ stability). • Enzymatic reaction mechanisms are similar to known organochemical mechanisms, BUT - they exhibit substrate ____________ - they operate billions of times faster - they can be regulated • Most are proteins. Why? They're more _______ than RNA. • Only a few are RNA molecules Why? They're left over from early evolution - slower and less easily regulated. Example: Ribosome catalyzes peptide bond formation.

all; reactant; specificity; flexible;

Protein Misfolding Diseases: - An ____________ is defined as any disorder, in which a protein forms ________ aggregates within/outside cells, impairing function - often with devastating effects. - Discovered by Stanley Prusiner (Nobel Laureate, 1997) Bucked idea that these diseases were from slow viruses Stubbornly isolated the first infectious protein

amyloidosis; insoluble

Starch: - made up of _________ (unbranched) and __________ (highly branched) forms of (alpha 1-4)-linked poly D-glucose - starch is enzymatically broken down from the _________________ ends to produce glucose for energy production

amylose; amylopectin; non-reducing

Naming Disaccharides: 1) start with configuration of ______ carbon joining left, ______________ monosacc. first to right, __________ monosacc. second 2) Name first residue with D/L and its ring structure 3) in parenthesis, indicate location of __________ bond 4) Do the same for the next sugar if it has 3 or more units

anomeric; non-reducing; reducing; glycosidic

Antibody structure is stabilized by -S-S- bonds: - Ab held together by intra-chain and inter-chain disulfides - without -S-S- linkages, a) Ab could not bind to _____ b) Ab would _________ and would be squeezed out of circulation c) Ab could not cross-link and ________ antigen-bearing cells

antigen; dissociate; agglutinate

Spectrometry: - Beer lambert law: Abs = ecl - abs = absorbance - e = molar absorptivity - l = cuvette length - c = ???? - Spectrophotometery allows us to detect the presence of _______- molecules and to _________ their concentration

aromatic; quantify

How Enzymes Promote Catalysis: - Orientation - _________ active-site for optimal activity - Induced-fit - binding substrate in ways that stabilize ________ and _________________ intermediates of catalysis - Acid-base catalysis - donating/abstracting _________ - Covalent catalysis - forming adducts or ____________ - Metal ion catalysis - exploiting the Lewis acid and _____ properties of active-site - Conformational catalysis - creating a ________ environment that facilitates chemical changes during _______ - Transition-state stabilization - ALL of the above, working together to _______ the activation energy for each step in a catalytic mechanism

arranging; chemical; conformational; protons; intermediates; redox; dynamic; catalysis; lower

Prolyl peptide bonds are different: • Cyclic side-chain reduces the energetic _______ to adopting the cis configuration. • Proline most often adopts the ____ configuration. - Has profound implications in the folding of a polypeptide into a fully functional protein. - By adopting cis configuration, prolyl residues cannot fit into a-helix. As such, they are ______ _________ that help many polypeptides adopt compact and stable spherical shape.

barrier; cis; helix terminators

Arrhenius Rate Theory: - explains reaction rate in terms of reaction ________ height - rate constant, k, depends on _____ ______ ___ ___________ - the higher delta G is, the ______ the reaction because the denominator will get exponentially ________ - as delta G gets bigger, k gets exponentially ________ - 10^13 is the ________ _______ of bond stretching which is the limit on how fast a reaction can go

barrier; free energy of activation; slower; bigger; smaller; vibrational frequency

Principal Buffers in Humans: - __________ - orthophosphate - atp - proteins Bicarbonate Buffer System - Arguably the most important buffer system for acid-base homeostasis in humans. - Takes full advantage of metabolically produced CO2. - Maintains a relatively constant plasma pH by countering changes in acids, bases, anions, metal ions, etc. - CO2 (_____) combines with water to form H2CO3 (_____), which then dissociates to form hydrogen ion & HCO3 - (CB) - When blood has excess hydrogen ions (__________), these protons shift the equilibrium in favor of CO2 & H2O, which is breathed out, thereby reducing body acidity.

bicarbonate; LA; BA; acidosis

- as water freezes, it expands, ripping plants apart, allowing C atoms to re-enter _____ ______ - h2o+co2 -> carbs is __________ - carbs -> metabolites is _______ - metabolites to Co2 is __________ - low bp relative to earths _________ - evaporative heat loss - keeps atmosphere moist - __________ remodels earth's landscape, increasing surface area and exposing mineral nutrients

carbon cycle; photosynthesis; anabolism; catabolism; temperature; erosion

Simple Enzymatic Reaction: - ES and EP are thermodynamic complexes, as defined by: KS = [E][S]/[ES] KP = [E][P]/[EP] - (EX) is the "joint transition-state", meaning that enzyme & substrate _______ simultaneously, remaining conformationally __________ to each other throughout catalysis. - (EX) is transient, lasting only a few picoseconds. - Think of the active site as a dynamic catalyst that promotes reactivity by adjusting the chemical environment by ________ _________

change; complementary; changing conformation

Covalent Catalysis: - A covalent bond forms transiently between enzyme and substrate - helps to preserve ________ _____ _______ during catalysis - AA Side-chains Modified: _____ of Lys, _______ of His, ______ of Cys, _______ of Asp, ________ of Thr and Ser

chemical bond energy; amino; imidazole; sulfur; carboxylate; hydroxyl

Starting with a protein of unknown sequence: -Most proteins have 400-500 amino-acid residues, Not possible to chemically sequence the entire protein. Must therefore cleave protein into smaller fragments. - We can illustrate the principles with a 28-residue peptide: - Treat separate samples of pure peptide with Trypsin, Chymotrypsin, or CNBr. - Isolate each fragment by ____________. - Chemically _________ each fragment.

chromatography; sequence

Salt Bridges: Bonds between oppositely charged residues that are sufficiently _______ to each other to experience mutual electrostatic attraction.

close

Properties of monosaccharides: - ___________ crystalline solid - freely soluble in _______ but insoluble in __________ solvents because of many hydroxyl groups - unbranched carbon chains

colorless; water; nonpolar

Equilibrium Constants & Rate Constants: • Equilibrium constant ( K'eq ) - defined by the relative __________ of substrates and products present at equilibrium in a biochemical reaction. - _____ value depends on standard free energy change • Rate constant ( k ) - proportionality constant that measures how _____ a particular reaction occurs. • Velocity has units of (M/s) • For first-order rxn (v depends only on [S] or [P]) • Rate of forward reaction: vforward = ____________ [S] • Rate of reverse reaction: vreverse = kreverse [P] - Rate is expressed by reaction velocity:

concentrations; Keq; fast; Kforward [S]; v = k [S]

Without catalyst, it's not as simple as it looks. Why? - H2O crowds around nucleophiles and electrophiles, thereby __________ nucleophilic attack. - Thermal agitation (diffusion) prevents optimal _________ of orbitals needed for most efficient nucleophilic attack. - The oxy-anion is a very ________ intermediate - Hard to have both base and acid in very same solution. - Even slower at room temperature and neutral pH

deactivating; alignment; unstable

- water crowds electron rich and electron deficient areas thereby __________ them - While seemingly disadvantageous, this effect is actually extremely beneficial & a key to Life. Why? - Uncatalyzed reactions are extremely slow under physiologic conditions (salty H2O, neutral pH, ambient temperature, atmospheric pressure) - Requires Enzyme Catalysis (________ & controllable) - example: glucose ________ with ____________ - Catalyzed reaction reaches equilibrium within ________, whereas uncatalyzed reaction reaches equilibrium in ___________≈ 30 years. - HK specifically phosphorylates at position-__, with no action at other hydroxyl groups. - Hexokinase is also _______ by glucose-6-P

deactivating; specific; phosphorylation; hexokinase; 1 sec; 1 billion sec; 6; regulated

Hydrophobic interactions stabilize proteins: - Strength of hydrophobic interaction is determined by number of organized water molecules released - oily groups form more ________ environments - Hydrophobic interactions also play a dominant role in stabilizing membrane _______.

decreases; neutral; bilayer

Pi-Cation Interactions: - Aromatic groups have highly ________ p-electron orbitals above and below plane of ring - These orbitals often interact with ___________ ammonium ions to form stable bonds that are important in enzyme-substrate interactions and receptor-agonist interactions. - In aqueous media, the cation-π interaction is comparable to (and potentially stronger than) _______________ salt bridges. - Cation-π interactions can tune pKa of nitrogenous sidechains, increasing the abundance of the ____________ form. - Nicotinic acetylcholine receptor (nAChR) which binds its endogenous ligand, acetylcholine (a positively charged molecule).

delocalized; quaternary; ammonium-carboxylate; protonated

Noncompetitive inhibition (reversible) - Inhibitor & substrate binds at _______ sites - Raising [S] cannot fully ______ inhibition. - Makes noncompetitive inhibitors _______ drugs.

different; reverse; effective

Reversible & Irreversible inhibitors: - Enzymes can be inhibited by small molecules (metabolic feedback inhibitors, _____, toxins, poisonous metals). - Bind to the active or inhibitor site, but don't form product. - Reversible Inhibitors: (associate/dissociate readily) • most bind ___________: E + I ⇌ EI • many compete ______ with substrate binding • others bind at another site that interferes with _______ • some bind extremely _______: E + I ⇌ EI1 ⇌ EI2 ⇌ EI3 ⇌ etc. (boa constrictor analogy) - Irreversible Inhibitors (forms a _______ bond)

drugs; reversibly; directly; catalysis; tightly; covalent;

So how does an enzyme help? - Recognizes and binds only certain _______, preventing unwanted hydrolysis of other esters (substrate specificity or substrate selectivity) - Uses substrate binding energy to remove H2O (called ________), thereby facilitating nucleophilic attack - Optimizes nucleophile-electrophile orbital alignment, greatly improving in-line ________ of leaving group - Changes its conformation during catalysis, thereby __________ catalytic acid or base groups to achieve greatest catalytic performance - Stabilizes the oxy-anion by positioning _____ of nearby peptide bonds that H-bond to oxy-anion, stabilizing it. - Reduces energetic barrier to product release from Enz•Product complex

esters; desolvation; displacement; repositioning; N-H

Features of well-folded proteins: - Most are ________ (spherical), thereby maximizing number of hydrophobic groups within the interior and giving great ________. - They are stable in physiologic fluids (e.g., aqueous solution (pH 7.2), with moderate salt, metal ions, etc.) - They sediment uniformly in an ______________ (400,000 x g). - They have well-defined _________ that is predictable on the basis of molecular weight, size and shape. - They show __________ evidence of a well-folded structure, as detected in NMR, ESR, UV/Visible spectra. - They can arrange themselves into well-defined ________ that are suitable for X-ray crystallography. - AND, they are biologically active (i.e., displaying specific ______ interactions and/or catalytic activity).

globular; stability; ultracentrifuge; diffusivity; spectroscopic; crystals; binding

Glycogen: - is the primary source of ________ in the body - Polymer of __________-linked D-glucose, with _______ branching every 8-12 residues - glycogen provides long-term ________ ________ in animals and fungi - is synthesized on a protein anchor that localizes it within ________

glucose; alpha 1-4; alpha 1-6; energy storage; cytoplasm

- __________ is any molecule possessing an O-, N-, or S- glycosidic bond - glycoside bonds involve _________ carbons (others are just simple _____) - They are stable ___________ and unstable ___________ - ____________ and some proteins have n-glycosidic bonds

glycoside; anomeric; ethers; kinetically; thermodynamically; nucleosides

Uncatalyzed Chemical Reaction: • S and P must surmount an energy barrier (ΔG‡ > 0), for a reaction to occur. • Equilibrium constant between S and P reflects the difference in the stability of their ______ states (ΔG'o). - Barrier height reflects the energy needed for: - __________ of molecular orbitals - _________ of electronic charges - bond ____________ - other transformations (protonation/deprotonation)

ground; alignment; separation; rearrangements

- If protein folding was a two state process: the barrier height would be too ______ and rate too ____ - it is a linear multi-step process: there are too many ______ and rate would be too _____

high; low; barriers; low

2 Proteins play crucial roles in cells: - Structural elements - collagen, __________, keratins. - Enzymes - catalysts for metabolism & ________ - Antibodies - immune surveillance - Receptors & Channels - signal transduction - Transporters - __________ import/export - Molecular Motors - ______ generation - Transcription Factors - gene ______ - Proteins are long polypeptides. Range in molecular weight from 5,000 to >1,000,000 - Most proteins are spherical (globular) These polypeptides fold to form compact 3-D structures that are required for biological activity. - Intrinsically Disordered Proteins Some proteins are structureless, but gain regular 3-D structure, but after binding to an well-folded protein.

histones; signaling; metabolite; force; control

Polysaccharides (glycans): - amylose = unbranched ________ of glucosyl units - ____________ contain a single type of monosacc. and are used for _____ ______ as well as ___________ - ______________ contain two or more varieties of monosacc. and provide _________ support for microorganisms (peptidoglycans) - unlike _______, polysacc. have a variable molecular weight. We call them indefinite ________

homopolymer; homopolysacc.; energy storage; structure; heteropolysacc. extracellular; proteins; polymers

- maltose -> alpha-D-glucose and beta-D-glucose is a _______ reaction - alpha-D-glucose and beta-D-glucose -> Maltose is a _______ reaction - maltose still possesses a _____ end

hydrolysis (add water); condensation (remove water); reducing

- in reality, there is no free H+, they transfer to a neighboring h2o, thereby forming ____________ - in ___________ a proton is transferred between 2 identical molecules: one H2O acts as a Brønsted acid (proton _______) and the other H2O molecule acts as a Brønsted base (proton acceptor).

hydronium; donor

Chaperones 16 Facilitate Protein Folding: - Some proteins have difficulty folding, thereby exposing their _________ interior - Chaperonins - proteins that "assist" unfolded proteins to fold into their biologically active structure Also prevent/suppress improper protein __________. Some chaperonins are _______________ that use ATP to exert molecular-scale forces to promote folding.

hydrophobic; aggregation; mechanoenzymes

The water cycle (or hydrologic cycle) - refers to the continuous exchange of water within the __________ - Exchange between the atmosphere, soil water, surface water, groundwater, plants & animals - __________ - from oceans and other water bodies into the atmosphere. - _____________ - from plants and animals into atmosphere. - ______________ - from water vapor condensing and falling to earth. - ________ - eventually reaching the sea.

hydrosphere; Evaporation; Transpiration; Precipitation; Runoff

Enzymatic Catalysis: - Notice that the enzyme has no effect on the _____ ____ _______ ______ of reaction (i.e., Keq is unchanged) - Enzymes enhance reaction rate by lowering the activation energy

initial and final states

Pauling's a-Helix (first observed in a-_____): - Helix is a combination of rotation & _________ - 3.6 residues per turn - 100 degree rotation per residue - H-bonding (III) is __________ within in the a-helix - Linus Pauling noticed the N-H of 1st residue gets very close to the O of of ____ residue - Francis Crick noticed that the a-helix ______ slightly

keratin; translation; maximized; 4th; curls

Cyanogen bromide (CNBr) fragmentation: - Cleaves only after* Met to produce two peptides-- one with homoserine ________ at its C-terminus - CNBr has NO effect when Methionine is on _________

lactone; C-terminus

- water has a ________ dipole movement - water is so ________ that interactions between H2O molecules are strong- giving it one of the highest _____ ______ of any known substance - lifetime of a H bond is in ________ - in liquid water, H bonds are relentlessly made and broken, liquid water is thus a flickering collection of ____ with extremely little if any, W1

large; polar; heat capacities; picosec; w5

a _________ H bond is stronger than _______

linear; bent

- Locke and Key mechanism not valid because ES complex would be stuck at ____ energy since the [ES] is bound to tightly making it harder for [ES] to get over energy barrier for _______ ______

low; product formation

- conversion between anomers can happen spontaneously in water via __________ which is when the ring opens and recloses - at equilibrium, about ___ is alpha-D-glucose and _____ B-D-glucose, with very small amounts of linear and the ______________ forms

mutarotation; 1/5; 4/5; glucofuranose

van der Waals interactions: - Short-lived bond resulting from interactions of fluctuating dipoles of ______________ electrons. - When time-averaged, the net effect is a weak bond. - van der Waals forces operate over very _____ distances (1/r3 ´ 1/r3) for two spherical electrostatic fields - Water interferes with van der Waals interactions. However, within a protein's ________ interior, numerous van der Waals interactions contribute considerably to protein stability. - usually __________ a.a's are involved

non-bonding; short; dehydrated; aromatic

Tertiary structures are stabilized by short-range _________ interactions: - Although disulfide bonds are useful in stabilizing some proteins, most protein structures lack _______ bonds or any other interchain covalent crosslinks. - Instead, local noncovalent forces stabilize the native structure including: ____________ interactions electrostatic interactions van der Waals interactions - These short-range (Ångström-scale) forces are strongest when water is ________. - Noncovalent interactions make it easier to disrupt structure, when time comes to remove old proteins.

noncovalent; -S-S-; hydrophobic; excluded; disrupt

Managing Binding Energy for Substrate Specificity and Effective Catalysis: • Binding Energy (DGB < 0) is derived from __________ enzyme-substrate interactions which is stabilizing. • Enzymes use binding energy: (1) for substrate specificity (______ amount of energy) (2) to harness molecular forces needed to stabilize transition state (E∙X)‡ (biggest amount of energy) • Substrate specificity is mainly achieved by exploiting ________ bonding, ionic interactions, ________ interactions, and van der Waals forces. • Noncovalent enzyme interactions in (E∙X)‡ are stronger than in _____ complex. • These non-covalent interactions also allow the enzyme to _______ the transition-state.

noncovalent; little; biggest; hydrogen, hydrophobic; E+S; stabilize

- alpha has OH on ____ side of ring as C6 (CH2OH) - Beta has OH on _____ side of ring as C6 - ____ determines D and L whereas the _______ carbon is where alpha and beta are - open chain aldehyde reacts with ____ groups in hemoglobin in diabetes to form _______

opposite; same; C5; anomeric; amino; imines

Monosaccharide Stereochemistry: - simple sugars have chiral centers and are therefore _______ ______ - fischer projection - horizontal bonds project ____ the plane and vertical bonds project _____ the plane - perspective formula - solid wedges project ____ plane and dashed wedges project ____ plane - n chiral centers = 2^n _____________ - sugars differing in configuration at only one carbon are called ______

optically active; out; in; out; in; stereoisomers; epimers

- One H2O molecule is lost in forming a _______ bond. - Product is called a peptide. What remains of each AA is called a _______. - N-terminus (written on left) & C-terminal (written on right) are unmodified -NH2 & -COOH In cells, peptide formation is catalyzed by ___________: tRNA molecules carry amino acids to the ribosome. Amino acids one-at-a-time, as directed by mRNA template - Peptides can also be made in the lab but we have to protect the ________ _____ - total number of possible sequences = _____, n= number of _______ - groups within a peptide _______ ionize

peptide; residue; ribosomes; side chains; 20^n; residues; cannot

Roles of Carbohydrates: how are carbs made? ______________ Carbs can provide energy by the oxidation of ______ and ______ Carbs can provide structure via __________ in the ECM Glycoconjugates (sugars linked to other metabolites) _________ and __________ membranes due to their ability to be incorporated into lipids and proteins

photosynthesis; sugar; starch; cellulose; stabilize; regulate

Hydrophobicity facilitates protein folding: - Protein folding decreases local entropy; however, water release increases entropy. So overall DS is ________. - Side-chains participating in hydrophobic interactions can ______, thereby facilitating protein folding - Great variety of hydrophobic side-chains maximizes packing & maximizes release of bound water. aka ________ A.A's

positive; swivel; nonpolar

In these days of easy DNA sequencing, one may ask "Why bother with fragment sequencing?" - REMEMBER: We're most often interested in the sequence of the physiologically active form of the protein. (prot. sequencing) - Many genes give rise proteins of different AA sequences, depending on the tissue in which they are expressed. - mRNA can be spliced, removing certain sequences and altering the actual sequence of the expressed protein. - Many proteins are ____________ modified to form specialized amino acids (e.g., _____________ in collagen; g-carboxyglutamate in blood clotting enzymes, etc.) - Many proteins are post-translationally cleaved, such that the final amino acid sequence does not match sequence encoded in the _____ _________. - Best to do both protein & DNA sequencing

post-translationally; hydroxyproline; DNA sequence

Turnover Number & Maximal velocity: - Turnover Number or kcat (=k3) is defined as the number of _________ molecules formed by each enzyme ______ site per second. - Vm = kcat [E]total - Think of kcat as the ________ of catalysis (i.e., number of catalytic cycles per second). - The value of kcat is a measure of _________ __________. Fastest enzymes have ________ kcat values. - Only kcat is a true constant. Its value only depends on an enzyme's _______ power. - Note that Vm CANNOT be a true constant because it depends on ______

product; active; frequency; catalytic efficiency; highest; [E]tot

- pH meters don't measure [H+]. - They measure ______ _______ (H+), where A is the activity coefficient, such that (H+) = A[H+]. - BUFFERS: Weak Acids & Weak Bases help to stabilize pH. They obey Le Chatelier's Principle: "a dynamic system ____________ to a stress"

proton activity; compensates

Ionization of Amino Acids: - Charge on amino acid depends on solution pH. - We begin at low pH (fully ________) and add OH- (removing acidic protons first) - the _________ acids have the weakest proton affinity and lose their H first - the strongest bases have _________ proton affinity and lose their H last

protonated; strongest

Ramachandran Plots Help to Define the Allowable Protein Conformational Space: - Main chain torsion angles _____ & ____ are relatively free to rotate, but side-chains can cause steric hindrance. - Ramachandran used models of small polypeptides to vary phi and psi systematically to identify stable conformations that are representative of each type of secondary structure - Ramachandran plot therefore represents the allowable ______ _______ for each residue within a protein. - energetically unfavorable steric clashes are defined by _____ areas

psi; phi; torsion angles; white

Kinetic investigation of reaction mechanisms: - Goal: To understand an enzyme's mechanism by measuring how ___________ _______ changes as a function of various experimental parameters (e.g., [S], [E], pH, [Metal Ion], [Coenzyme], etc.). - Detect all reaction __________ to define the chemistry. - Measure ____ effects to reveal role of acid/base catalysis. - Assess how ___________ structure affects reaction rates. (specificity) - Determine how site-directed ________ of enzyme affects reaction rates. (Tells us about role of active-site residues.) - Define the _______ of the steps in multi-substrate reactions. (type of mechanism) - Determine how _________ molecules alter catalysis (helps learn control)

reaction rate; intermediates; pH; substrate; mutation; order; regulatory

Stability of Folded Proteins: Folding of a protein, and hence its conformational stability, depends on the sum of DH and DS for three factors: 1. Protein folding necessarily _______ local entropy: -TDSfolding > 0 (_______ structural organization) 2. Net favorable enthalpy from ___________ interactions: DH < 0 (reflects a favorable change in heat capacity) where DH H-bonding = 0 (no net energy advantage) DH ionic < 0 (salt bridge energy release) DH hydrophobic > 0 DH van der Waals < 0 3. Major contribution from the favorable _________ in entropy of water release attending burying hydrophobic residues -TDS hydrophobic-effect << 0 (randomness of H2O release)

reduces; higher; intramolecular; increase

Competitive inhibition (__________) - Inhibitor binds __ ________ of substrate - raising [S] fully reverses ________ because the inhibitor will be outcompeted. - makes for poor drugs

reversible; in place; inhibitor

Protein synthesis & folding occur on virtually the same timescale: - Electron micrograph shows polypeptides are already folding, well before they are released from ____________ _________.

ribosome/mRNA complex

Two Ideas about Protein Folding: In one model, folding occurs in stages: - Nucleation - formation of local __________ structures (e.g., a-helices and b-sheets), leading to folding __________ - Structural _____________ of intermediate states - continued formation of secondary structure and beginnings of tertiary structure to give higher level intermediate structures - Final rearrangements - once the basic elements in place, final adjustments result in a fully ________ protein - In second model, folding is initiated by rapid collapse into a more _______ state - Formation of compact state is largely driven by _________ interactions - Compact state called "molten globule", implying a fluid (conformationally _______) condition. - Molten globule favors massive ____ search for stability. - Folding is most likely a ____ of both ideas.

secondary; intermediates; consolidation; functional; compact; hydrophobic; supple; local; mix

`Roles of Weak Noncovalent Interactions: - 5 basic types - though individually weak, their overall effect can be very ___________ - allows proteins and nucleic acids to fold and unfold on biology time-scale - hydrogen bonds are between ________ (and polar??) groups and peptide bonds - ionic attractions include attraction and repulsion - hydrophobic interactions - van der Waals interactions

significant; neutral;

Polysacc. Branching: - Sugars are very water _____ due to large number of ___ groups - By storing glucose in ______ or ________, free glucose concentrations are kept down, as is ____________ - ___________ is flexible and much more _______

soluble; OH; starch; glycogen; osmolarity; amylopectin; soluble

How Proteins Fold Remains a Mystery: - Solution conditions (e.g., water, pH, salt, temperature, metal ions, etc.) strongly influence structural _______. - RNA & Proteins are stabilized by _________ Interactions, H-Bonding, Electrostatics & van der Waals Interactions. Same forces most likely drive folding of RNA & proteins. - Levinthal Paradox - Cyrus Levinthal noted that the very _______ number of degrees of freedom in an unfolded polypeptide means it has an _________ number of possible conformations. If folding were to occur as a series of individual steps, the folding time for a 600- residue protein would exceed the age of the Earth!! - SO HOW CAN IT WORK? Levinthal predicted that only a massively _______ search of local conformation space would permit a polypeptide to reach a stable and active structure on a biologically relevant timescale.

stability; Hydrophobic; large; astronomical; parallel

Hydrophobic Interactions: (main driving force for ___________) - Strictly speaking, hydrophobic interactions are NOT ______ _____ (There are NO atomic or molecular orbitals.) - The Gibbs Equation (DG = DH - TDS) is guiding: - Contrary to intuition, water binds fairly well to ________ groups, such that: DHwater-__________ << 0 DHwater-___________ >> 0 - Hydrophobic (nonpolar??) interaction releases many H2O molecules, greatly increasing _________

stabilization; true bonds; apolar; binding; release; randomness

Globular proteins exhibit well-defined biological activity: - Specific Binding is an intensive variable that is defined as mol _________ (aka ligand) bound per mol _______. (usually one binding site on each polypeptide chain). - Example: Intact antibody binds 2 mol antigen Fab fragment binds 1 mol antigen - Specific Activity is an intensive variable defined as µmol reaction _________ formed per minute per mg enzyme. (measure enzyme purity in terms of biological purity) - 1 mg pure yeast hexokinase catalyzes the formation of 172 µmol glucose-6-P per minute

substance; protein; product

Uncompetitive inhibition (reversible) - ________ binding creates a site for inhibitor binding - ____ inhibitory mode because once we form ES, it doesn't stay in that conformation for long - with ________ is reversible - No effect of inhibitor on ______

substrate; Rare; dilution; slope

Enzyme Kinetics: - Best way to identify physiologically relevant _________. - Best way to define an enzyme's _________ mechanism. - Best way to understand an enzyme's role within a _________ pathway. - Best way to discover ________ activators & inhibitors. - Best way to develop _____. - Best way to pinpoint the biochemical _______ of disease (e.g., to identify a mutation's effect on catalysis). .

substrates; catalytic; metabolic; allosteric; drugs; basis

Electrostatic Interactions: delta E= fd - E repulsion - For charges of same sign, MORE energy must be _________ to hold A and B near each other. - E attraction (fav) - for charges of __________ sign, energy is released spontaneously, and A-B bond is stable. - Ionic groups tend to be on the ______ of proteins. - Formation of ionic bond _______ water

supplied; opposite; surface; releases

Design of enzyme transition-state inhibitors: - Rationale: Substances resembling the reaction's ________ state should bind far more ________ than the substrate, - - - - They are already ________ distorted to resemble T-state - Approach: We use enzyme _______ to infer the geometry of the most likely _______ state, and we then synthesize a chemically stable analogue of the transition state. - Why not synthesize the actual transition-state? - TS is very _____ and _______ molecule

transition; tightly; sterically; kinetics; transition; fast; unstable

Noncovalent Interactions Play Key Roles in Living Systems: - While organic chemistry focuses on covalent bond rearrangements, living systems are highly dependent on both covalent bonds and noncovalent interactions. - Unlike the considerable bond energies (≈ 350-500 kJmol) of single, double and triple bonds, the strengths of noncovalent bonding interactions are "________" - meaning their weaker energies depend on many factors, such as hydration, ___________, polarity, etc. - Noncovalent interactions are made and/or broken readily. - Noncovalent interactions determine protein structure and function; _________ __________; DNA structure and function; membrane formation and stability; and nearly every other vital process in living systems.

tunable; electrostatics; enzyme specificity

Cellulose: - an ____________ polymer of D-glucose units that are connected by ____1-4 glycosidic bonds - extensive _______________ H-bonding as well as H-bonding between ________ chains - most animals lack enzymes that can cleave cellulose

unbranched; beta; intramolecular; cellulose

- hydrolysis is defined as the breaking apart of molecules by H2O. - Ionic hydrolysis (_________) - Covalent hydrolysis (enzyme-catalyzed) - the two examples of enzymes in covalent hydrolysis are: ___________ and glucose 6-phosphotase

uncatalyzed; carbonic anhydrase

Cysteine & Cystine: - Free SH group in cysteine ionizes (pKa = 8.3) and is largely _________ at physiologic pH - SH groups __________ to form disulfide bond in cystine: - Cystine is mainly present in __________. (Note: In most cases, the cytoplasm is reduced, whereas extracellular regions are oxidized.)

undissociated; oxidize; circulation

Protein Refolding: - Proteins can be denatured (unfolded, with loss of activity) - Occurs upon exposure to organic solvents, _____, ________, and heat - Some proteins can be renatured (i.e., can refold into active (Native) three-dimensional structure) - Anfinsen showed ribonuclease can be unfolded & refolded. - Proved that primary sequence of polypeptide predicts 3-d structure (Nobel Prize, 1972) Even so, we still cannot predict how a protein ______,based only on its sequence. - Because RNAse is an enzyme, Anfinsen used its catalytic activity to quantify the extent of __________.

urea; detergents; folds; refolding

Why should we care about enzyme reaction mechanisms? • To understand how enzymes ______ • To understand what happens when enzymes don't work (i.e., the origin of numerous diseases/disorders).(can fix them) • To understand how enzymes can increase reaction rates so dramatically. • To understand the basis of substrate specificity - the key to forming _________ pathways • To understand how to _______ enzymes in the design of highly effective drugs, pesticides,herbicides, etc.).

work; metabolic; inhibit