Biochemistry 1995,34, 117 13- 11724
11713
Structure-Reactivity Relationships for P-Galactosidase (Escherichia coli, lac 2). 2. Reactions of the Galactosyl-Enzyme Intermediate with Alcohols and Azide Ion? John P. Richard,*-$John G. Westerfeld,g Sue Lin,# and James Beards." Department of Chemistry, University at Buffalo, SUNY, Buffalo, New York 14260-3000, and Department of Chemistry, University of Kentucky, Lexington, Kentucky 40506-0055 Received May 5, 1995; Revised Manuscript Received July 11, 1995@
ABSTRACT: Velocities for the synthesis of P-D-galactopyranosyl derivatives by transfer of the galactosyl group from P-galactosidase to seven alkyl alcohols, glucose, and azide ion have been determined as the difference in the velocities for P-galactosidase-catalyzed cleavage of 4-nitrophenyl P-D-galactopyranoside to give 4-nitrophenoxide anion (VPNP) and hydrolysis of this compound to give D-galactose ( ~ ~ 1 ) Rate . constant ratios kROH/k, (M-l) for partitioning of the galactosylated enzyme between reaction with alkyl alcohols and solvent determined by this method are in good agreement with values of k ~ o ~ (M-l) / k ~ determined by analysis of alcohol inhibition of enzyme-catalyzed hydrolysis of the corresponding alkyl P-D-galactopyranosides. Absolute rate constants k ~ (M-I o ~s-') for reaction of alkyl alcohols with the galactosylated enzyme intermediate were calculated from the corresponding rate constant ratio kROH/ks (M-l) and k, = 710 s-l. A Brgnsted parameter of = -0.19 f 0.10 was determined from the second-order rate constants for the reactions of alcohols with the galactosylated enzyme. The large difference between @lg)kCat,k;, = -0.75 k 0.14 for cleavage of alkyl P-D-galactopyranosides to form the galactosylated enzyme and = -0.19 for the reverse synthesis reaction requires that the equilibrium constants for galactosyl group transfer from alkyl P-D-galactopyranosides to the enzyme increase sharply 0.05 for the with decreasing pK, of the alkyl alcohol leaving group. These data give Peq = -0.56 reaction of alkyl P-D-galactopyranosideswith ethanol to form ethyl P-D-galactopyranosideand alkyl alcohol. Several effects that lead to this increased ease of cleavage of alkyl P-D-galactopyranosides with decreasing basicity of the alkoxy group are discussed. A second-order rate constant of kcilC = 1.2 x lo4 M-' s-l was determined for reaction of glucose with the galactosylated enzyme. The relatively low reactivity of glucose is surprising, because an earlier observation that the galactosylated enzyme complex generated by the cleavage of lactose undergoes release of glucose and synthesis of allolactose at nearly equal rates suggests that the binding of glucose to the galactosylated enzyme should be partly irreversible and that it takes place near the encounter-controlled limit. The data suggest a significant stabilization of nonproductive complexes formed by binding of glucose to the galactosylated enzyme. P-Galactosidase catalyzes the hydrolysis of P-D-galactopyranosyl azide, but not the synthesis of this compound by reaction of azide ion with the galactosylated enzyme. This suggests that different forms of P-galactosidase catalyze the cleavage and synthesis of P-D-galactopyranosyl azide. This may correspond to a change in the state of ionization of the residue that participates in acid-base catalysis of the reaction in the cleavage and synthesis directions.
vnuc)~0~
vnuc)~0~
,&Galactosidase catalyzes the hydrolysis of lactose and other P-D-galactopyranosyl derivatives by a two-step mechanism through a galactosyl-enzyme reaction intermediate. The large secondary a-deuterium isotope effect of kdkD = 1.25 (Sinnott & Souchard, 1973) for the enzyme from Escherichia coli (lac Z ) on the rate constant k, (s-l) for transfer of the galactosyl group from enzyme to water shows that there is a large change from sp3 to sp2 hybridization at carbon- 1 of the galactosyl moiety on moving to the transition state for hydrolysis of the intermediate, which requires sp3hybridized covalent attachment of the intermediate to the enzyme. The point of covalent attachment of the galactosyl + This work was supported by Grant GM 47307 from the National Institute of General Medical Sciences. * Address correspondence to this author. t University at Buffalo, SUNY. University of Kentucky. Present address: Department of Chemistry, Catawba College, Salisbury, NC 28144-2488. Abstract published in Advance ACS Abstracts, September 1, 1995. @
0006-2960/95/0434-11713$09.OO/O
group was identified as Glu-537, by degradation and amino acid analysis of a stable 2-deoxy-2-fluoro-~-~-galactopyranosy1 enzyme intermediate generated by enzyme-catalyzed cleavage of the corresponding 2,4-dinitrophenyl P-D-galactopyranoside (Gebler et al., 1992). The absolute rate constant of k, = 1300 s-' for transfer of the galactosyl group to water at 25 "C and pH 7 was determined as the turnover number k,,, when hydrolysis of the intermediate is rate-determining for reaction of /3-D-galactopyranosyl derivatives (Sinnott & Souchard, 1973). These data show that P-galactosidase causes a profound change in the reactivity of the acylal linkage, the cleavage of which is more than 108-fold faster than that of simple glycosidic acylals in water (Brown & Bruice, 1973). The reaction of the galactosyl-enzyme intermediate in water containing 1 M methanol results in a 70% yield of methyl P-D-galactopyranoside (Viratelle & Yon, 1973; Sinnott & Viratelle, 1973). A rate constant ratio of kMeoH/ks = 2.2 M-' for partitioning of the intermediate, and an
0 1995 American Chemical Society
11714 Biochemistry, Vol. 34, No. 37, 1995
Richard et al.
Scheme 1
constant KT = (kcat/Km)/kROH for transfer of the galactosyl group from alkyl P-D-galactopyranosides to the enzyme with decreasing basicity of the alkoxy leaving group, which is correlated by PT = -0.56. This is a surprising result with important consequences for the interpretation of these Bransted parameters. There is no detectable trapping of the galactosyl-enzyme intermediate by the very good nucleophile azide ion at concentrations of up to 0.5 M. The observation of enzymatic catalysis of the cleavage, but not the synthesis, of P-Dgalactopyranosyl azide has interesting implications with respect to the nature of the interactions between the enzyme catalyst and anionic nucleophilesAeaving groups.
absolute rate constant kMeOH = 2900 M-' s-l for its reaction with methanol, were calculated from these results (Sinnott & Viratelle, 1973). In this paper, we expand upon the methanol trapping experiments and report the results of a study of the effect of changing the pKa of the alcohol nucleophile on the reactivity of the alcohol toward the galactosyl-enzyme intermediate and an attempt to trap the intermediate with the highly reactive nucleophile azide ion. These experiments were designed to address several questions. (1) How does the value of the Bransted coefficient Bnuc for transfer of the galactosyl group from the enzyme to external alcohols compare with related values determined for nucleophilic substitution reactions in water, which proceed by well-defined chemical mechanisms (Jencks & Jencks, 1977; Jencks, 1985; Richard & Jencks, 1984a; TaShma & Jencks, 1986)? This comparison may exclude some mechanisms for the galactosyl transfer reaction and provide information about the nature of the rate-determiningtransition state. (2) What is the effect of changing alkoxy substituents on the equilibrium constants for transfer of the galactosyl group from alkyl P-D-galactopyranosides to P-galactosidase? These equilibrium constants may be calculated from a Haldane relationship for reversible cleavage and formation of alkyl P-D-galactopyranosides: KT = (kcat/Km)/kROH (Scheme 1A), using kinetic data for the cleavage reaction reported in the previous paper (Richard et al., 1995). (3) Does P-galactosidase catalyze the transfer of the galactosyl group to nucleophilic anions? P-Galactosidase is a reasonably efficient catalyst of cleavage of 8-Dgalactopyranosyl azide (Sinnott, 1971), and a superficial application of the principal of microscopic reversibility would appear to require that the enzyme also catalyze the reverse nucleophilic reaction of azide ion with the galactosylenzyme intermediate (Scheme 1B). We report here the effect of changing pKa on the secondorder rate constants for the reaction of short-chain alkyl alcohols with the galactosyl-enzyme intermediate of the P-galactosidase-catalyzed cleavage of 4-nitrophenyl p-Dgalactopyranoside. There is a modest increase in kRoH (M-' s-l) with decreasing alcohol pK,, which is correlated by the Bransted parameter Pnuc = -0.19 f 0.10. This is consistent with a mechanism involving general-base catalysis of the addition of these alcohols to an oxocarbenium ion reaction intermediate. By contrast, a significantly more negative value of = -0.75 f 0.14 was determined for the enzyme-catalyzed cleavage of alkyl p-D-galactopyranosides, so that there must be a large increase in the equilibrium
MATERIALS AND METHODS The sources for most of the chemicals and enzymes used in this work are given in the preceding paper (Richard et al., 1995). p-D-Galactopyranosyl azide was synthesized by a published procedure (Sinnott, 1971). Methyl B-D-galactopyranoside and D-galactose were purchased from Sigma and used without further purification. Deuterium oxide (99.9% D) and methanold4 (99.8% D) were purchased from Cambridge Isotope Laboratories. 'H NMR spectra at 400 MHz were recorded in D20 on a Varian VXR-400 spectrometer. Chemical shifts are reported relative to HOD at 6 = 4.65 ppm. Enzyme Assays. The methods for routine assays of the activity of P-galactosidase and galactose dehydrogenase were described in the preceding paper (Richard et al., 1995). Magnesium-free P-galactosidase was prepared by extensive dialysis against 10 mM EDTA, and the magnesium-free enzyme was assayed in the presence of 10 mM EDTA (Tenu et al., 1972). P-Galactosidase-catalyzed hydrolysis of P-Dgalactopyranosyl azide was monitored by coupling the formation of D-galactose to the reduction of NAD+ using galactose dehydrogenase (Richard et al., 1995). The reaction of 2-nitrophenyl P-D-galactopyranosidein the presence of increasing concentrations of trifluoroethanol was monitored by following the formation of 2-nitrophenoxide anion ( k 4 1 0 = 4300 M-' cm-', pH 8.6) in a solution containing 25 mM sodium pyrophosphate (pH 8.6), 1.0 mM MgC12, and 2.2 mM 2-nitrophenyl P-D-galactopyranoside at 25 "C. p-Galactosidase-catalyzed hydrolyses of fixed concentrations of alkyl P-D-galactopyranosides in the presence of increasing concentrations of the corresponding alkyl alcohol leaving group were monitored by the galactose dehydrogenase-coupled enzyme assay (Richard et al., 1995). The following concentrations of alkyl P-D-galactopyranosides were used for these experiments: ethyl P-D-galactopyranoside, 1.43 mM; 2-chloroethyl P-D-galactopyranoside, 0.033 mM; trifluoroethyl P-D-galactopyranoside, 0.042 mM. The initial velocities of formation of 4-nitrophenoxide anion and of D-galactose in the P-galactosidase-catalyzed cleavage of 4-nitrophenyl P-D-galactopyranoside were determined at 25 "C in a single-assay solution that contained 25 mM sodium pyrophosphate (pH 8.6), 1.0 mM MgC12, 0.7 mM NAD+, 0.5 mM 4-nitrophenyl P-D-galactopyranoside, and 0.3 unit of galactose dehydrogenase in a total volume of 1.0 mL. The absorbance at 340 nm was monitored until it was constant with time (-3 min), and
Galactosyl Transfer to Nucleophiles P-galactosidase was then added. The formation of Dgalactose was monitored for 4 min by following the increase in absorbance at 340 nm (hA340), and the formation of 4-nitrophenoxide anion was then monitored for 4 min, by following the increase in absorbance at 405 nm (AA405). Control experiments showed the following: (1) Identical results were obtained when h A 4 0 5 was monitored for 4 min first, followed by &4340. (2) There is no significant change in absorbance at 405 nm associated with the reduction of NAD+. (3) The change in absorbance at 340 nm due to the formation of 4-nitrophenoxide is 25-fold smaller than the change in absorbance at 405 nm. The initial velocity (
