Time to Connect……. Enzyme Activity is amount of enzyme that converts one micromole of substrate to product in one minute Velocity is micromoles product formed per minute This slide is intended to show the connection between enzyme activity and Vmax. Vmax is maximum product formed at saturating substrate per minute for a given amount of enzyme,

Km meaures the affinity of the enzyme for the E + S [ES] E + P There are 2 steps in an enzyme-catalyzed reaction 1. Formation of ES Km 2. Formation of P kcat Km meaures the affinity of the enzyme for the substrate. It does not concern product formation Kcat measures how rapidly the product is formed from ES. It does not measure binding of substrate

Time to Connect……. Vmax = kcat [E] Kcat = k2 when ES = ET or Vmax Therefore: Vmax = kcat [E] or Kcat = Vmax [E] mmoles S per minute mmoles Enzyme = Turnover kcat is synonymous with Turnover Number

Multiple the numerator by the reciprocal of the denominator Time to Get Efficient……. At maximum efficiency, k1 controls the rate k2 k2 + k-1 k1 kcat / Km = Multiple the numerator by the reciprocal of the denominator Every collision results in ES k1k2 k2 + k-1 = k2 When an enzyme is maximally efficient, k2 >>k-1 k1 = kcat / Km of 108 or 109 means diffusion control

kcat kcat/Km Km(M) Chymotrypsin 0.14 9.3 Urease 0.025 1 x 104 4 x 105 (Per second) Chymotrypsin 0.015 0.14 9.3 Urease 0.025 1 x 104 4 x 105 Acetylcholine esterase 0.000095 1.4 x 104 1.5 x 108 Catalase 0.025 1 x 107 4 x 108 The rate of catalysis by Acetylcholine esterase and catalase is controlled by diffusion … Every collision with the enzyme results in a product

Metal Ions in Catalysis- One third of all enzymes require a metal ion for catalysis

Zn 2+Polarizes H2O, making it a better nucleophile His –Zn2+ His O H .. + C His –Zn2+ His O H C H2O His –Zn2+ His O H .. + H+ + H C Bicarbonate Displaces HCO3-

RNA Hydrolysis . .. O O-P-O-CH2 Base O-P H CH2 O O-P-O-CH2 Base H N P + .. 2’,3’-cylic nucleotide N . H2O His 12 O Base O-P CH2 H O H N H + His 119

RNA Hydrolysis (cont.) : = 3’ end of split RNA O O-P-O-CH2 Base O O N His 12 O P = O O H 3’ end of split RNA H N + His 119 + N

Insights into the Mechanism of a Digestive Enzyme 1. Some digestive enzymes catalyze the hydrolysis of proteins. Examples are: trypsin and chymotrypsin 2. Some have a Serine residue at the catalytic site 3. Histidine and Aspartate are also needed 4. The 3 make up a catalytic triad

Catalytic Triad of Chymotrypsin : Histidine C O CH2 CH HN O=C : H O CH2 C= N CH .. Attacking Group on Serine Aspartate Catalytic Site Serine

Chymotrypsin Hydrophobic Pocket O C Asp N H His Ser .. HO—C — CH— NH—C —CH —NH —A-A-A-A-A-A-A-A- O R Hydrophobic Pocket

Chymotrypsin Split Protein O C Asp N H His Ser C —CH —NH —A-A-A-A-A-A- AA—C — CH—NH3 O R

Chymotrypsin Split Protein O C Asp N H His Ser H O : C —CH —NH —A-A-A-A-A-A Split Protein O AA—C — CH—NH3 O R

Chymotrypsin Broken Peptide Bond O C Asp N H His Ser H C —CH —NH —A-A-A-A-A-A O O AA—C — CH—NH3 O R

-chymotrypsin (active) -chymotrypsin (active) Zymogens Rule: Many hydrolytic enzymes are synthesized as inactive precursors. The suffix “ogen”, or the prefix pro, and prepro” designate a precursor Rule: Activation requires removal of a blocking peptide by proteolytic cleavage Trypsin COOH S - S Chymotrypsinogen (inactive) COOH S - S -chymotrypsin (active) Chymotrypsin COOH S - S -chymotrypsin (active)

Hexokinase Random More than One Substrate….. E + S1 + S2 E + S1 then S2 E-S1-S2 E + P1 + P2 E + S2 then S1 Hexokinase Example Glucose + ATP E + Glucose E-Glucose + ATP or E-Glucose-ATP E + ATP E-ATP + Glucose

ORDERED More than One Substrate….. E + S1 + S2 E + S1 E-S1 E-S1-S2 No Reaction Example Alcohol Dehydrogenase CH3CH2OH + NAD+ CH3CHO + NADH + H+ Enz + NAD+ Enz-NAD+ Enz-NAD+ + CH3CH2OH Enz-NAD+ CH3CH2OH

PING PONG P1 P2 S1 S2 E ES1 E* E*S2 E First product appears before second substrate binds Serine Proteases use a Ping Pong Mechanism