Ground-State, Transition-State, and Metal-Cation Effects of the 2-Hydroxyl Group on -D-Galacto
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- 作者：John P. Richard ; Deborah A. McCall ; Christina K. Heo ; and Maria M. Toteva
- 刊名：Biochemistry
- 出版年：2005
- 出版时间：September 6, 2005
- 年：2005
- 卷：44
- 期：35
- 页码：11872 - 11881
- 全文大小：132K
- 年卷期：v.44,no.35(September 6, 2005)
- ISSN：1520-4995
文摘
Substitution of the C2-OH group by C2-H at 4-nitrophenyl-
-D-galactopyranoside to give4-nitrophenyl-2-deoxy--D-galactopyranoside causes (1) a change in the rate-determining step for-galactosidase-catalyzed sugar hydrolysis from formation to breakdown of a covalent intermediate; (2)a 14 000-fold decrease in the second-order rate constant k3/Kd for enzyme-catalyzed transfer of the -D-galactopyranosyl group from the substrate to form a covalent adduct to the enzyme; and (3) a larger320 000-fold decrease in the first-order rate constant ks for hydrolysis of this covalent adduct. Only asmall fraction (ca. 7%) of the 2-OH substituent effect is expressed in the ground-state Michaelis complex,so that the (apparent) strong interactions between the enzyme and 2-OH group that stabilize the transitionstate for -D-galactopyranosyl transfer only develop upon moving from the Michaelis complex to thetransition state. Mg2+ activates -galactosidase for cleavage of both 4-nitrophenyl--D-galactopyranosideand 4-nitrophenyl-2-deoxy--D-galactopyranoside. This suggests that Mg2+ activation does not involveinteractions with the 2-OH group. The removal of Mg2+ from -galactosidase causes a change in therate-determining step for enzyme-catalyzed hydrolysis of 4-nitrophenyl-2-deoxy--D-galactopyranosidefrom breakdown to formation of the covalent intermediate. The observed 2-OH effect would require avery large (10-11 kcal/mol) stabilization of the transition state for -D-galactopyranosyl group transferto water by interactions between -galactosidase and the neutral 2-OH group. We suggest that the apparenteffect of the neutral substituent is more simply rationalized by ionization of the 2-OH to form a 2-O-anion, which provides effective electrostatic stabilization of the cationic transition state for glycosidecleavage at an active site of relatively low dielectric constant.
-D-galactopyranoside to give4-nitrophenyl-2-deoxy--D-galactopyranoside causes (1) a change in the rate-determining step for-galactosidase-catalyzed sugar hydrolysis from formation to breakdown of a covalent intermediate; (2)a 14 000-fold decrease in the second-order rate constant k3/Kd for enzyme-catalyzed transfer of the -D-galactopyranosyl group from the substrate to form a covalent adduct to the enzyme; and (3) a larger320 000-fold decrease in the first-order rate constant ks for hydrolysis of this covalent adduct. Only asmall fraction (ca. 7%) of the 2-OH substituent effect is expressed in the ground-state Michaelis complex,so that the (apparent) strong interactions between the enzyme and 2-OH group that stabilize the transitionstate for -D-galactopyranosyl transfer only develop upon moving from the Michaelis complex to thetransition state. Mg2+ activates -galactosidase for cleavage of both 4-nitrophenyl--D-galactopyranosideand 4-nitrophenyl-2-deoxy--D-galactopyranoside. This suggests that Mg2+ activation does not involveinteractions with the 2-OH group. The removal of Mg2+ from -galactosidase causes a change in therate-determining step for enzyme-catalyzed hydrolysis of 4-nitrophenyl-2-deoxy--D-galactopyranosidefrom breakdown to formation of the covalent intermediate. The observed 2-OH effect would require avery large (10-11 kcal/mol) stabilization of the transition state for -D-galactopyranosyl group transferto water by interactions between -galactosidase and the neutral 2-OH group. We suggest that the apparenteffect of the neutral substituent is more simply rationalized by ionization of the 2-OH to form a 2-O-anion, which provides effective electrostatic stabilization of the cationic transition state for glycosidecleavage at an active site of relatively low dielectric constant.