title: Structure-Reactivity Relationships for β-Galactosidase (Escherichia coli, lac Z): A Second Derivative Effect on β(nuc) for Addition of Alkyl Alcohols to an Oxocarbenium Ion Reaction Intermediate.
abstract: Velocities for the synthesis of trifluoroethyl 2-deoxy-β-D-galactopyranoside by transfer of the 2-deoxygalactosyl group from β-galactosidase to trifluoroethanol were determined from studies of the β-galactosidase-catalyzed cleavage of 4-nitrophenyl-2-deoxy-β-D-galactopyranoside as the difference in rates of appearance of 4-nitrophenoxide anion and 2-D-deoxygalactose. These data were used to calculate a rate constant ratio of k(ROH)/k(s) = 2.3 M(-1) for partitioning of the intermediate between addition of trifluoroethanol and solvent water. Velocities for the synthesis of other alkyl 2-deoxy-β-D-galactopyranosides by transfer of the 2-deoxygalactosyl group from β-galactosidase to alkyl alcohols were determined from the effect of alkyl alcohols on the velocity of β-galactosidase-catalyzed cleavage of 4-nitrophenyl-2-deoxy-β-D-galactopyranoside in a reaction where breakdown of the intermediate is rate determining. These data were used to calculate rate constant ratios k(ROH)/k(s) for the reactions of eight alkyl alcohols. Absolute rate constants k(ROH) (M(-1) s(-1)) were calculated from k(ROH)/k(s) and k(s) = 0.002 s(-1) for the addition of water. A Brønsted coefficient of β(nuc) = -0.07 ± 0.08 was determined as the slope of a logarithmic correlation of k(ROH) against alcohol pK(a). The change from a 2-OH to a 2-H substituent at the β-D-galactopyranosyl intermediate causes a 0.12 ± 0.04 increase in the value of β(nuc) for alcohol addition. This anti-Hammond effect provides evidence that general basecatalyzed addition of alcohols to an enzyme bound β-D-galactopyranosyl oxocarbenium ion intermediate proceeds along a reaction coordinate in which there is strong coupling between carbon-oxygen bond formation and proton transfer from the alcohol to a basic residue at the enzyme.
authors.affiliation: Contribution from the Department of Chemistry, University at Buffalo, SUNY, Buffalo, New York 14260-3000.
authors.forename: John P
authors.initials: JP
authors.lastname: Richard
authors.forename: Christina K
authors.initials: CK
authors.lastname: Heo
authors.forename: Maria M
authors.initials: MM
authors.lastname: Toteva
references.citation: Biochemistry. 1999 Sep 14;38(37):12111-23
references.pmid: 10508416
references.citation: Biochem J. 1973 May;133(1):81-7
references.pmid: 4721624
references.citation: Science. 1989 Mar 17;243(4897):1485-8
references.pmid: 2538921
references.citation: J Am Chem Soc. 2004 Sep 29;126(38):11814-9
references.pmid: 15382915
references.citation: Biochemistry. 2002 Mar 5;41(9):3207-25
references.pmid: 11863460
references.citation: Biochemistry. 2005 Sep 6;44(35):11872-81
references.pmid: 16128589
references.citation: Biochemistry. 1996 Sep 10;35(36):11797-804
references.pmid: 8794761
references.citation: Carbohydr Res. 2000 Jul 10;327(1-2):27-46
references.pmid: 10968675
references.citation: Biochemistry. 1974 Nov 5;13(23):4715-20
references.pmid: 4609464
references.citation: Biochemistry. 1995 Sep 19;34(37):11703-12
references.pmid: 7547902
references.citation: Biochem J. 1974 Dec;143(3):751-62
references.pmid: 4462753
references.citation: Biochem J. 1973 May;133(1):89-98
references.pmid: 4578762
references.citation: Biochemistry. 1996 Sep 24;35(38):12387-401
references.pmid: 8823174
references.citation: Biochemistry. 1998 Mar 31;37(13):4305-9
references.pmid: 9556344
references.citation: Biochemistry. 1995 Sep 19;34(37):11713-24
references.pmid: 7547903
references.citation: Biochem J. 1971 Dec;125(3):717-9
references.pmid: 4947657
references.citation: Biochemistry. 1996 Sep 24;35(38):12377-86
references.pmid: 8823173