CHMI E.R. Gauthier, Ph.D. 1 CHMI 2227E Biochemistry I Enzymes: - catalysis

CHMI E.R. Gauthier, Ph.D.2 Enzyme catalysis Upon binding to the enzyme’s active site, the substrate is surrounded by several side chains that:  Hold the substrate in place;  Participate in the reaction.

CHMI E.R. Gauthier, Ph.D.3 Enzyme catalysis Basic reaction mechanisms  General acid/base catalysis:  Involves the transfer of protons between side chains of the enzyme and the substrate;  The most common form of enzyme catalysis Three main types of catalysis are found:  Covalent catalysis:  Involves the formation of a transient covalent bond between the enzyme and substrate;  This covalent bond activates the substrate for further reaction ;  Metal ion catalysis:  Several enzymes use metal ions for catalysis (Mg +2, Mn +2, Zn +2, Fe +2, Cu +2 );  The metal ion can act is different ways:  Stabilises the transition state;  Helps to orientate the substrate vs the enzyme;  Participates in the transfer of electrons/protons between the enzyme and the substrate;

CHMI E.R. Gauthier, Ph.D.4 Enzyme catalysis General acid/base catalysis Chymotrypsin O OH R HOR + H

CHMI E.R. Gauthier, Ph.D.5 Enzyme catalysis Metal ion catalysis H+H+

CHMI E.R. Gauthier, Ph.D.6 Enzyme catalysis Covalent catalysis Nucleophile

CHMI E.R. Gauthier, Ph.D.7 Enzyme catalysis Refresher – nucleophiles and electrophiles

CHMI E.R. Gauthier, Ph.D.8 Enzyme catalysis - Enzymes often combine two catalytic strategies General Acid/ base catalysis Covalent catalysis

CHMI E.R. Gauthier, Ph.D.9 Example of enzyme catalysis 1. Chymotrypsin Protease:  Cuts peptide bonds C-ter of Tyr/Phe/Trp Serine protease:  Ser 195 actively participates in the catalysis Catalytic triad:  His 57, Asp1 02, Ser 195

CHMI E.R. Gauthier, Ph.D.10 Example of enzyme catalysis 1. Chymotrypsin

CHMI E.R. Gauthier, Ph.D.11 Example of enzyme catalysis Importance of the catalytic triad The determine the importance of the catalytic triad in catalysis, each amino acid of the triad of the protease subtilisin were changed to alanines; Note: the Y axis is a Log scale.

CHMI E.R. Gauthier, Ph.D.12 Example of enzyme catalysis 1. Chymotrypsin - specificity

CHMI E.R. Gauthier, Ph.D.13 Example of enzyme catalysis 1. Chymotrypsin - specificity Ser 195

CHMI E.R. Gauthier, Ph.D.14 Example of enzyme catalysis 1. Chymotrypsin - catalysis Catalytic triad Substrate C-ter part of the protein N-ter part of the protein Peptide bond cleavage Enzyme regeneration Covalent intermediate

CHMI E.R. Gauthier, Ph.D.15 Example of enzyme catalysis 1. Chymotrypsin - catalysis The oxyanion hole is formed by Ser 195 and Gly 193 ; The formation of H- bonds between the peptide bond NH group of these amino acids and the O - stabilizes the reaction intermediate

CHMI E.R. Gauthier, Ph.D.16 Example of enzyme catalysis Similarities between different proteases

CHMI E.R. Gauthier, Ph.D.17 Example of enzyme catalysis Similarities between different proteases

CHMI E.R. Gauthier, Ph.D.18 Example of enzyme catalysis 2. Enolase Enolase catalyses one of the multiple enzymatic reactions of glycolysis (the degradation of glucose into pyruvate);

CHMI E.R. Gauthier, Ph.D.19 Example of enzyme catalysis 2. Enolase

CHMI E.R. Gauthier, Ph.D.20 Example of enzyme catalysis 2. Enolase

CHMI E.R. Gauthier, Ph.D.21 Example of enzyme catalysis 3. Lysozyme Lysozyme is present in tears and egg white; It degrades sugars found on the bacterial cell wall (peptidoglycan);

CHMI E.R. Gauthier, Ph.D.22 Example of enzyme catalysis 3. Lysozyme Sugar portion of peptidoglycan

CHMI E.R. Gauthier, Ph.D.23 Example of enzyme catalysis 3. Lysozyme

CHMI E.R. Gauthier, Ph.D.24 Example of enzyme catalysis 3. Lysozyme H2OH2O 2