Chymotrypsin胰凝乳蛋白酶 catalysis CH3 N-terminus C-terminus NH2 H3C 0 0 +H3N HO Ala Phe Asn Ser Met Glu Figure 9-1 Biochemistrv.Sixth Edition Figure 9.1 Specificity of chymotrypsin.Chymotrypsin cleaves proteins on the carboxyl side of aromatic or large hydrophobic amino acids. 裂解芳香氨基酸或大的疏水氨基酸（桔色）C-端肽键 (红色)
Figure 9.1 Specificity of chymotrypsin. Chymotrypsin cleaves proteins on the carboxyl side of aromatic or large hydrophobic amino acids. Chymotrypsin 胰凝乳蛋白酶 catalysis N-terminus C-terminus 裂解芳香氨基酸或大的疏水氨基酸（桔色）C-端肽键（红色）
Chymotrypsin Possesses a Highly Reactive Serine Residue 高度活泼的Ser残基 Chymotrypsin is a good example of the use of covalent catalysis.共价修饰 A powerful nucleophile Ser to attack the unreactive carbonyl carbon atom of the substrate. CH +F+H Serine 195 Ser 195 与抑制剂结合 CH CH DIPF Figure 9.2 An unusually reactive serine in chymotrypsin
Figure 9.2 An unusually reactive serine in chymotrypsin. Chymotrypsin Possesses a Highly Reactive Serine Residue 高度活泼的Ser残基 Chymotrypsin is a good example of the use of covalent catalysis. 共价修饰 - A powerful nucleophile Ser to attack the unreactive carbonyl carbon atom of the substrate. Serine 195 与抑制剂结合
Chromogenic substrate for enzyme activity detection N-Acetyl-L-phenylalanine p-nitrophenyl ester +H20= p-Nitrophenolate Figure9-3 Biochemistry,Sixth Edition 2007W.H.Freeman and Company Figure 9.3 Chromogenic substrate.N-Acetyl-L-phenylalanine p- nitrophenyl ester yields a yellow product,p-nitrophenolate,on cleavage by chymotrypsin
Figure 9.3 Chromogenic substrate. N-Acetyl-L-phenylalanine pnitrophenyl ester yields a yellow product, p-nitrophenolate, on cleavage by chymotrypsin. Chromogenic substrate for enzyme activity detection
Chymotrypsin:a Covalently Bound Intermediate 酶催化包括共价结合中间物 酶动力学：Kvof20mM Kcat of 77 s-1. The initial phase of the reaction was examined by using the stopped-flow method,which makes it possible to mix enzyme and substrate and monitor the results within a millisecond. 截留分光光度法检测反应的起始状态，能够在毫秒范围内混合 酶和底物、跟踪毫秒时间内的反应结果。 Steady-state phase Two stages a rapid burst phase(pre-steady state)and a steady-state phase. 前稳态与稳态过程 Burst phase Milliseconds after mixing-> irstt
Two stages： a rapid burst phase (pre-steady state) and a steady-state phase. 前稳态与稳态过程 The initial phase of the reaction was examined by using the stopped-flow method, which makes it possible to mix enzyme and substrate and monitor the results within a millisecond. 截留分光光度法检测反应的起始状态，能够在毫秒范围内混合 酶和底物、跟踪毫秒时间内的反应结果。 Chymotrypsin: a Covalently Bound Intermediate 酶催化包括共价结合中间物 酶动力学： KM of 20 mM kcat of 77 s - 1
The formation of a covalently bound ES intermediate This nucleophile becomes covalently attached to the substrate briefly in the course of catalysis.亲核基团短暂地 结合到底物上 酰化酶中间物水解， 底物的酰基与酶共价 释放底物的羧酸组分 结合，同时释放一个 和游离酶 产物 Fast Slow (A) (B) Acylation Deacylation OH X OH HO R XH H20 XH=ROH(ester), RNH2(amide) Enzyme Acyl-enzyme Enzyme Figure9-5 Biochemistry,Sixth Edition acyl-enzyme intermediate 2007 W.H.Freeman and Company 酰化酶中间物
底物的酰基与酶共价 结合，同时释放一个 产物 The formation of a covalently bound ES intermediate acyl-enzyme intermediate 酰化酶中间物 Fast Slow This nucleophile becomes covalently attached to the substrate briefly in the course of catalysis. 亲核基团短暂地 结合到底物上 酰化酶中间物水解， 释放底物的羧酸组分 和游离酶