Enzymes Most biological catalysts are proteins (some REALLY COOL ONES are folded RNAs!!!) Catalysts - change rate of reaction without net change of catalyst Catalyst does not alter equilibrium Enzyme Nonenzymatic reaction rate (s -1 ) Enzymatic reaction rate (s -1 ) Rate enhancement Carbonic anhydrase 1.3 x x x 10 6 Triose phosphate isomerase 4.3 x x 10 9 Staphlococcal nuclease 1.7 x x 10 14

Enzymes E + SESE + P Highly specific Reaction occurs in active site of enzyme Substance acted upon = substrate Resulting species = product Enzyme acts on forward and reverse reactions Activity depends on protein’s native structure Regulated - by concentrations of substrate and substances other than substrate

Enzymes Cofactors/Coenzymes Functional groups of protein enzymes are involved in acid-base reactions, covalent bond formation, charge-charge interactions BUT they are less suitable for oxid-reduc and group-transfer reactions SO they use COFACTORS (inorganic ions) COFACTORS may be metal ions (Cu 2+, Fe 3+, Zn 2+ ) trace amounts of metal needed in our diets

COFACTORS can be organic or metalloorganic molecules --> COENZYMES Examples: NAD + Heme Enzymes Cofactors/Coenzymes Holoenzyme = Apoenzyme (inactive) + cofactor/coenzyme/metal ions

Enzymes Coenzymes Coenzymes must be regenerated Many vitamins are coenzyme precursors Vitamins must be present in our diets because we cannot synthesize certain parts of coenzymes CoenzymeReaction mediatedVitamin source Human Disease Cobalamin coenzymes AlkylationCobalamin (B12)Pernicious anemia Flavin coenzymes Oxidation-reductionRiboflavin (B2)rare Nicotinamide coenzymes Oxidation-reductionNicotinamide (niacin)Pellagra Pyridoxal phosphate Amino group transferPyridoxine (B6)rare TetrahydrofolateOne-carbon group transfersFolic acidMegaloblastic anemia Thiamine pyrophosphate Aldehyde transferThiamine (B1)Beriberi

Enzymes Substrate specificity Types of complementarity between enzyme and substrate: Geometric Electronic Hydrophobic Hydrophilic Substrate binding sites undergo conformational change when substrate binds induced fit “lock-and-key”

Enzyme undergoes conformational change when substrate binds - induced fit Enzymes a b c Substrate + Enzyme a b c ES complex a b c

Enzyme-substrate complementarity Dihydrofolate reductase-NADP + (red)-tetrahydrofolate (yellow)

Enzymes Stereospecific Why? Inherently chiral (proteins only consist of L-amino acids) so form asymmetric active sites Example: Protein enzyme Yeast Alcohol dehydrogenase (YADH) CH 3 CH 2 OH + NAD + YADH CH 3 CH + NADH + H + O EthanolAcetaldehyde

Enzymes Stereospecific Yeast Alcohol dehydrogenase (YADH) is stereospecific 1. If YADH reaction uses deuterated ethanol, NAD + is deuterated to form NADD N H + R C O NH 2 + CH 3 CD 2 OH (ethanol) YADH + CH 3 CD + H + O N H R C O NH 2 NAD + D NADD N H R C O NH 2 D 2. Isolate NADD and use in reverse reaction to reduce normal acetaldehyde, deuterium transferred from NADD to acetaldehyde to form ethanol + CH 3 CH + H + O YADH CH 3 C D pro-R H pro-S OH +NAD + 3. Enantiomer of ethanol - none of deuterium is transferred from this isomer of ethanol to NAD + in the reverse reaction CH 3 C H pro-R D pro-S OH (acetaldehyde)

Enzyme activity Dependent on: [metal ion], pH, temperature, [enzyme], [substrate]

Enzymes E + SESE + P  G’˚ < 0; favorable

Enzymes Enzymes affect reaction rates, not equilibria Catalysts enhance reaction rates by lowering activation energy Rate is set by activation energy  G ‡ Higher activation energy --> slower reaction Overall rate of reaction is determined by step with highest activation energy --> rate-limiting step

General acid catalysis - partial proton transfer from an acid lowers free energy of reaction’s transition state Enzymes General acid-base catalysis General base catalysis - partial proton abstraction by a base lowers free energy of reaction’s transition state KetoTransition stateEnol R C CH 2 H O H A R C CH 2 H O HA     R C CH 2 OH A- H KetoTransition stateEnol R C CH 2 H O R C H O    R C CH 2 OH H B H+ B B 

Enzymes General acid-base catalysis

Example:Ribonuclease A (RNase A) digestive enzyme secreted by pancreas into small intestine hydrolyzes RNA rate depends on pH, suggesting involvement of ionizable residues His12 and His119 Enzymes General acid-base catalysis

Transient covalent bond formed between E and S Accelerates reaction rate through transient formation of a catalyst-substrate covalent bond Usually covalent bond is formed by the reaction of a nucleophilic group on the catalyst with an electrophilic group on the substrate --> nucleophilic catalysis Enzymes Covalent Catalysis H2OH2O S A -S B + N: S A -N + S B S A + N: + S B Example: Decarboxylation of acetoacetate (catalyst contains primary amine) CCH 3 CH 2 O- O + RNH 2 C O OH- CCH 3 CH 2 O- N C O R H + CCH 3 CH 2 N R H.. CO 2 CCH 3 N R H + + H + RNH 2 + OH- CCH 3 O acetoacetate acetone SCHIFF BASE (IMINE)

Some amino acids with nucleophilic groups ROHSerine RSHCysteine RNH 3 +Lysine Histidine NHHN + R Enzymes Covalent Catalysis

Enzymes Metal Ion Catalysis One-third of all known enzymes require metal ions --> metalloenzymes Fe 2+, Fe 3+, Cu 2+, Zn 2+, Mn 2+, Co 2+ (sometimes Na +, K +, Mg 2+, Ca 2+ ) Metal bound to enzyme (or substrate) What can it do? help orient substrate (or enzyme) for reaction stabilize charged reaction transition state mediate oxidation-reduction reactions (change metal’s oxidation state) Voet, p , scheme

Enzymes: Chymotrypsin Serine protease, very reactive serine residue in enzyme Digestive enzyme synthesized by pancreas Catalyzes cleavage of peptide bonds adjacent to aromatic amino acids Transition state stabilization General acid-base catalysis and covalent catalysis Catalytic triad = Ser 195, Asp 102, His 57

Enzymes: Chymotrypsin general base general acid general base general acid Covalent intermediate

Enzymes: Chymotrypsin

Enzymes: Chymotrypsin

Enzymes: Chymotrypsin

Enzymes: Chymotrypsin

Enzymes: Chymotrypsin and other serine proteases

Enzymes: Enolase catalyzes reaction step of glycolysis reversible dehydration of 2-phosphoglycerate to phosphoenolpyruvate Metal ion catalysis, general acid-base, transition state stabilization Lys 345 = general base, abstracts proton from C-2 of 2-phosphoglycerate Glu 211 = general acid, donates proton to -OH leaving group

Enzymes: Enolase Metal ion catalysis 2 Mg 2+ ions interact with 2-phosphoglycerate making the C-2 proton more acidic (lower pK a ) and easier to abstract