Enzyme Catalysis: Structural Basis and Energetics of Catalysis

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PHRM 836 September 8, 2015

Enzyme Catalysis: structural basis and energetics of catalysis

Devlin, section 10.3 to 10.5 1. Enzyme binding of substrates and other ligands (binding sites, structural mobility) 2. Energecs along reacon coordinate 3. Cofactors 4. Eﬀect of pH on enzyme catalysis Enzyme catalysis: Review Devlin sections 10.6 and 10.7 • Deﬁnions of catalysis, transion state, acvaon energy • Michaelis-Menten equaon

– Kinec parameters in enzyme kinecs (kcat, kcat/KM, Vmax, etc) – Lineweaver-Burk plot • Transion-state stabilizaon • Meaning of proximity, orientaon, strain, and electrostac stabilizaon in enzyme catalysis • General acid/base catalysis • Covalent catalysis

2015, September 8 PHRM 836 - Devlin Ch 10 2 Structure determines enzymatic catalysis as illustrated by this mechanism for ____

2015, September 8 PHRM 836 - Devlin Ch 10 www.studyblue.com 3

Substrate binding by enzymes • Highly complementary interacons between substrate and enzyme – Hydrophobic to hydrophobic – Hydrogen bonding – Favorable Coulombic interacons • Substrate binding typically involves some degree of conformaonal change in the enzyme – Enzymes need to be ﬂexible for substrate binding and catalysis. – Provides opmal recognion of substrates – Brings catalycally important residues to the right posion.

2015, September 8 PHRM 836 - Devlin Ch 10 4 Substrate binding by enzymes • Highly complementary interacons between substrate and enzyme – Hydrophobic to hydrophobic – Hydrogen bonding – Favorable Coulombic interacons • Substrate binding typically involves some degree of conformaonal change in the enzyme – Enzymes need to be ﬂexible for substrate binding and catalysis. – Provides opmal recognion of substrates – Brings catalycally important residues to the right posion.

Induced ﬁt: promoted by rotation around Orotate Figure 10.11 bonds phosphoribosyltransferase

2015, September 8 PHRM 836 - Devlin Ch 10 5 Substrate binding by enzymes • Highly complementary interacons between substrate and enzyme – Hydrophobic to hydrophobic – Hydrogen bonding – Favorable Coulombic interacons • Substrate binding typically involves some degree of conformaonal change in the enzyme – Enzymes need to be ﬂexible for substrate binding and catalysis. – Provides opmal recognion of substrates – Brings catalycally important residues to the right posion. • Substrate binding site is a relavely small region Alcohol dehydrogenase (dimer) - Ethanol à acetaldehyde 2015, September 8 - NADH, Zn cofactors 6 Transion-state binding vs substrate binding • Enzyme must bind substrate, transion state and product. • Tight binding to substrate or product slows overall reacon by increasing the height of the barrier to TS* or product dissociaon, respecvely. • Tight binding to TS* speeds the reacon.

Figure 10.15

2015, September 8 PHRM 836 - Devlin Ch 10 7 Transion states • Rate enhancement of a chemical reacon by transion state stabilizaon. • Paral charges occur frequently in transion states.

Reactant Transion state Product chemical intermediate; very short-lived; high energy Figure 10.13

2015, September 8 PHRM 836 - Devlin Ch 10 8 Transion state stabilizaon: an illustraon

Arg 127

Environment optimal for reaction! Figure 10.39, 10.40 2015, September 8 PHRM 836 - Devlin Ch 10 12 Cofactors and coenzymes

1. Non-protein small molecules required for funcon of some enzymes 2. Organic cofactors are also called coenzymes or prosthec groups. – Many (not all) are derivaves of vitamins – For some enzymes, are chemically modiﬁed during the reacon – Funcon: hydride or electron transfer; group transfer 3. Inorganic cofactors – Metal ions – Metal clusters – Funcon: polarize bonds; coordinaon; metal reducon/ oxidaon

More informaon: hp://en.wikipedia.org/wiki/Cofactor_(biochemistry) 2015, September 8 PHRM 836 - Devlin Ch 10 13 Examples of important organic cofactors or coenzymes

More informaon: hp://en.wikipedia.org/wiki/Cofactor_(biochemistry) 2015, September 8 PHRM 836 - Devlin Ch 10 14 Textbook of Biochemistry with Clinical Correlaons, 7e edited by Thomas M. Devlin © 2011 John Wiley & Sons, Inc. Enzyme acve sites: cofactors bind as substrates

zinc

ethanol

NAD (analogue)

zinc

ethanol

NAD (analogue)

Alcohol dehydrogenase (dimer) Acve Site - Ethanol à acetaldehyde - Uses zinc and NAD hp://www.rcsb.org/pdb/101/motm_disscussed_entry.do?id=1adc#.Ujb9HgFU9us.email 2015, September 8 PHRM 836 - Devlin Ch 10 16 Eﬀect of pH on enzyme catalysis • When the rate-liming component of the catalyc process involves a tratable residue, the measured acvity of the enzyme depends on pH and the ionizaon status of that residue. à Highest enzymac acvity occurs with the proper ionizaon state. • Example below for general acid involvement in the catalyc step: – Enz—AH (acvite) ó Enz—A- (inacve)

– Enzyme acvity = 50% maximum acvity when pH = pKa of the general acid

1.0

0.8 pKa = ? 0.6 Is the proper ionizaon state 0.4 protonated or unprotonated? Relative Activity Relative 0.2

0.0 4 5 6 7 8 9 10 pH

2015, September 8 PHRM 836 - Devlin Ch 10 17 Eﬀect of pH on enzyme catalysis Example of pH dependence due to binding • Alcohol dehydrogenase (ADH) has 3 isoforms with diﬀerent pH opma • NADH release is the rate-liming step

ADH pH Res Res isoform opma 47 369 β1 10 Arg Arg β2 8.5 His Arg β2 7.0 Arg Cys Arg 47 Arg 369 Explain the basis for this pH dependence (Clinical Correlation 10.6)

2015, September 8 PHRM 836 - Devlin Ch 10 18 Summary of Enzyme Catalysis

• The funcon of enzymes is inmately linked to their structure – Speciﬁcity for substrate, cofactors (induced changes in structure) – Stabilizaon of the transion state, which deﬁnes enzyme catalysis • The pH dependence of catalysis derives from interacons of tratable groups formed in the rate- liming steps of the reacon mechanism, whatever that step may be (bond making/breaking; product release; substrate binding, etc).

2015, September 8 PHRM 836 - Devlin Ch 10 19