Enzymes mechanisms regulation Andy Howard Introductory Biochemistry 17

Enzymes: mechanisms & regulation Andy Howard Introductory Biochemistry 17 November 2014 Enzyme Mechanisms 11/17/2014

Enzyme mechanisms Many enzymatic mechanisms involve either covalent catalysis or acid-base interactions n We’ll give some examples of several mechanistic approaches n Then we’ll talk about enzyme activity is regulated n 11/17/2014 Enzyme Mechanisms P. 2 of 65

Mechanism Topics n n n Redox reactions Induced fit Ionic intermediates Active-site amino acids Serine proteases n n Reaction How they illustrate what we’ve learned Specificity Evolution 11/17/2014 Enzyme Mechanisms P. 3 of 65

Oxidation-Reduction Reactions n n Commonplace in biochemistry: EC 1 Oxidation is a loss of electrons Reduction is the gain of electrons In practice, often: n n oxidation is decrease in # of C-H bonds; reduction is increase in # of C-H bonds 11/17/2014 Enzyme Mechanisms P. 4 of 65

Redox, continued n n n Intermediate electron acceptors and donors are organic moieties or metals Ultimate electron acceptor in aerobic organisms is usually dioxygen (O 2) Anaerobic organisms usually employ other electron acceptors 11/17/2014 Enzyme Mechanisms P. 5 of 65

Biological redox reactions n n n n Generally 2 -electron transformations Often involve alcohols, aldehydes, ketones, carboxylic acids, C=C bonds: R 1 R 2 CH-OH + X R 1 R 2 C=O + XH 2 R 1 HC=O + X + OH- R 1 COO- + XH 2 X is usually NAD, NADP, FAD, FMN A few biological redox systems involve metal ions or Fe-S complexes Usually reduced compounds are higher-energy than the corresponding oxidized compounds 11/17/2014 Enzyme Mechanisms P. 6 of 65

One-electron redox reactions n n n FMN, FAD, some metal ions can be oxidized or reduced one electron at a time With organic cofactors this generally leaves a free radical in each of two places Subsequent reactions get us back to an even number of electrons 11/17/2014 Enzyme Mechanisms P. 7 of 65

Covalent catalysis n n Reactive side-chain can be a nucleophile or an electrophile; nucleophile is more common Bifidobacterium sucrose phosphorylase n A—X + E X—E + A EC 2. 4. 1. 7 n X—E + B B—X + E 113 k. Da dimer PDB 1 R 7 A, 1. 8Å Example: sucrose phosphorylase n n Net reaction: Sucrose + Pi Glucose 1 -P + fructose Fructose=A, Glucose=X, Phosphate=B 11/17/2014 Enzyme Mechanisms P. 8 of 65

Example: hexokinase Human brain Hexokinase I EC 2. 7. 1. 1 n Glucose + ATP Glucose-6 - + ADP 104 k. Da monomer n Risk: unproductive reaction with water PDB 1 CZA, 1. 9Å n Enzyme exists in open & closed forms P n n Glucose induces conversion to closed form; water can’t do that Energy expended moving to closed form 11/17/2014 Enzyme Mechanisms P. 9 of 65

Hexokinase structure n Diagram courtesy E. Marcotte, UT Austin 11/17/2014 Enzyme Mechanisms P. 10 of 65

Tight binding of ionic intermediates n n Quasi-stable ionic species strongly bound by ion-pair and H-bond interactions Similar to notion that transition states are the most tightly bound species, but these are more stable 11/17/2014 Enzyme Mechanisms P. 11 of 65

Reactive sidechains in a. a. ’s AA Group Charge @p. H=7 Asp —COO-1 Glu —COO-1 His Imidazole ~0 Cys —CH 2 SH ~0 Tyr Phenol 0 Lys NH 3+ +1 Arg guanidinium +1 Ser —CH 2 OH 0 Functions Cation binding, H+ transfer Same as above Proton transfer Covalent binding of acyl gps H-bonding to ligands Anion binding, H+ transfer Anion binding See cys 11/17/2014 Enzyme Mechanisms P. 12 of 65

Generalizations about activesite amino acids n n n Typical enzyme has 2 -6 key catalytic residues His, asp, arg, glu, lys account for 64% Remember: n n p. Ka values in proteins sometimes different from those of isolated amino acids Frequency overall Frequency in catalysis 11/17/2014 Enzyme Mechanisms P. 13 of 65

Rates often depend on p. H n n If an amino acid that is necessary to the mechanism changes protonation state at a particular p. H, then the reaction may be allowed or disallowed depending on p. H Two ionizable residues means there may be a narrow p. H optimum for catalysis 11/17/2014 Enzyme Mechanisms P. 14 of 65

Papain as an example 11/17/2014 Enzyme Mechanisms P. 15 of 65

i. Clicker quiz, question 1 1. Why would the nonproductive hexokinase reaction H 2 O + ATP ADP + Pi be considered nonproductive? n (a) Because it needlessly soaks up water n (b) Because the enzyme undergoes a wasteful conformational change n (c) Because the energy in the high-energy phosphate bond is unavailable for other purposes n (d) Because ADP is poisonous n (e) None of the above 11/17/2014 Enzyme Mechanisms P. 16 of 65

i. Clicker Quiz question 2 2. Triosephosphate isomerase (TIM) interconverts dihydroxyacetone phosphate (DHAP) and Dglyceraldehyde 3 -phosphate. What would bind tightest in the TIM active site? n (a) DHAP (substrate) n (b) D-glyceraldehyde (product) n (c) 2 -phosphoglycolate (Transition-state analog) n (d) They would all bind equally well n (e) None of them would bind at all. 11/17/2014 Enzyme Mechanisms P. 17 of 65

Serine protease mechanism n n n Only detailed mechanism that we’ll ask you to memorize One of the first to be elucidated Well studied structurally Illustrates many other mechanisms Instance of convergent and divergent evolution 11/17/2014 Enzyme Mechanisms P. 18 of 65

The reaction n n Hydrolytic cleavage of peptide bond Enzyme usually works on esters too Found in eukaryotic digestive enzymes and in bacterial systems Widely-varying substrate specificities n n Some proteases are highly specific for particular amino acids at position 1, 2, -1, . . . Others are more promiscuous O CH NH R 1 NH C O 11/17/2014 Enzyme Mechanisms C NH CH R-1 P. 19 of 65

Mechanism n n Active-site serine —OH … Without neighboring amino acids, it’s fairly unreactive becomes powerful nucleophile because OH proton lies near unprotonated N of His This N can abstract the hydrogen at nearneutral p. H Resulting + charge on His is stabilized by its proximity to a nearby carboxylate group on an aspartate side-chain. 11/17/2014 Enzyme Mechanisms P. 20 of 65

Catalytic triad n The catalytic triad of asp, his, and ser is found in an approximately linear arrangement in all the serine proteases, all the way from non-specific, secreted bacterial proteases to highly regulated and highly specific mammalian proteases. 11/17/2014 Enzyme Mechanisms P. 21 of 65

Diagram of first 3 steps (of 7) 11/17/2014 Enzyme Mechanisms P. 22 of 65

Diagram of last four steps Diagrams courtesy University of Virginia 11/17/2014 Enzyme Mechanisms P. 23 of 65

Chymotrypsin as example n n n Catalytic Ser is Ser 195 Asp is 102, His is 57 Note symmetry of mechanism: steps read similarly L R and R L Diagram courtesy of Anthony Serianni, University of Notre Dame 11/17/2014 Enzyme Mechanisms P. 24 of 65

Oxyanion hole n n When his-57 accepts proton from Ser-195: it creates an R—O- ion on Ser sidechain In reality the Ser O immediately becomes covalently bonded to substrate carbonyl carbon, moving negative charge to the carbonyl O. Oxyanion is on the substrate's oxygen Oxyanion stabilized by additional interaction in addition to the protonated his 57: main-chain NH group from gly 193 H-bonds to oxygen atom (or ion) from the substrate, further stabilizing the ion. 11/17/2014 Enzyme Mechanisms P. 25 of 65

Oxyanion hole cartoon n Cartoon courtesy Henry Jakubowski, College of St. Benedict / St. John’s University 11/17/2014 Enzyme Mechanisms P. 26 of 65

Modes of catalysis in serine proteases n n Proximity effect: gathering of reactants in steps 1 and 4 Acid-base catalysis at histidine in steps 2 and 4 Covalent catalysis on serine hydroxymethyl group in steps 2 -5 So both chemical (acid-base & covalent) and binding modes (proximity & transition-state) are used in this mechanism 11/17/2014 Enzyme Mechanisms P. 27 of 65

What mechanistic concepts do serine proteases not illustrate? n n Quaternary structural effects (We’ll discuss this under regulation…) Protein-protein interactions (Becoming increasingly important) Allostery (also will be discussed under regulation) Noncompetitive inhibition 11/17/2014 Enzyme Mechanisms P. 28 of 65

Specificity n n n Active site catalytic triad is nearly invariant for eukaryotic serine proteases Remainder of cavity where reaction occurs varies significantly from protease to protease. In chymotrypsin hydrophobic pocket just upstream of the position where scissile bond sits This accommodates large hydrophobic side chain like that of phe, and doesn’t comfortably accommodate hydrophilic or small side chain. Thus specificity is conferred by the shape and electrostatic character of the site. 11/17/2014 Enzyme Mechanisms P. 29 of 65

Chymotrypsin active site n n Comfortably accommodates aromatics at S 1 site Differs from other mammalian serine proteases in specificity Diagram courtesy chemistry program, Eastern Washington Univ. 11/17/2014 Enzyme Mechanisms P. 30 of 65

Divergent evolution n n Ancestral eukaryotic serine proteases presumably have differentiated into forms with different side-chain specificities Chymotrypsin is substantially conserved within eukaryotes, but is distinctly different from elastase 11/17/2014 Enzyme Mechanisms P. 31 of 65

i. Clicker quiz, question 3 3. Why are proteases often synthesized as zymogens? (a) Because the transcriptional machinery cannot function otherwise n (b) To prevent the enzyme from cleaving peptide bonds outside of its intended realm n (c) To exert control over the proteolytic reaction n (d) None of the above n 11/17/2014 Enzyme Mechanisms P. 32 of 65

i. Clicker question 4 4. Which of these enzymes would you predict to be the most similar to human pancreatic elastase? n n (a) human pancreatic chymotrypsin (b) porcine pancreatic elastase (c) subtilisin from Bacillus subtilis (d) none of these would be very similar to human pancreatic elastase 11/17/2014 Enzyme Mechanisms P. 33 of 65

Convergent evolution n n Reappearance of ser-his-asp triad in unrelated settings Subtilisin: externals very different from mammalian serine proteases; triad same 11/17/2014 Enzyme Mechanisms P. 34 of 65

Subtilisin mutagenesis n n n Substitutions for any of the amino acids in the catalytic triad has disastrous effects on the catalytic activity, as measured by kcat. Km affected only slightly, since the structure of the binding pocket is not altered very much by conservative mutations. An interesting (and somewhat non-intuitive) result is that even these "broken" enzymes still catalyze the hydrolysis of some test substrates at much higher rates than buffer alone would provide. I would encourage you to think about why that might be true. 11/17/2014 Enzyme Mechanisms P. 35 of 65