Biochemistry 0 zyxwvutsrqp Copyright 1978 by the American Chemical Society Volume 17, Number 20 Use of zyxwvuts pH Studies to Elucidate the Chemical Mechanism of Yeast Hexokinasef Ronald E. Viola and W. W. Cleland* ABSTRACT: The pH variation of the kinetic parameters of the reaction catalyzed by yeast hexokinase was examined for the forward and reverse reactions. In the forward direction, the kinetic parameters showed a decrease on the acid side below pH 7. The V, and V/K profile for MgATP had a “hollow” in the profiles, which was eliminated by the addition of citrate. The V/K profile for 2,5-anhydro-~-mannitol also gave a hollow in the profile, which was eliminated by the ad- dition of citrate; however, the V/K profile for 1,5-anhydro- D-mannitol did not have a hollow and was not activated by citrate. The V/K profile for glucose did not show this hollow and subsequent citrate activation, but reflects several groups whose protonation caused loss of activity. At least some of these groups are important for substrate binding, but not for catal- ysis, and cannot be protonated in the enzyme-glucose complex. T h e long-standing controversy concerning the kinetic mechanism of yeast hexokinase (EC 2.7.1.1) has apparently been resolved in favor of a random kinetic mechanism (Ru- dolph and Fromm, 1971; Danenberg and Cleland, 1975). Work is now proceeding in several labs to determine the individual steps in the chemical mechanism, the groups on the enzyme which are responsible for the binding of substrates, and the groups which participate in the catalytic steps. Chemical- modification studies have been carried out by several labs in an attempt to determine the amino acid residues essential for activity. Histidyl residues have been shown to have no role in either binding or catalysis (Grouselle et al., 1973), while evi- dence has been presented (Pho et al., 1974) to show the in- volvement of carboxyl groups at the active site. Later work led to the isolation of a modified glutamyl residue which was proposed to have a catalytic function (Pho et al., 1977). The modification of a single tyrosyl residue caused the loss of ac- tivity (Coffe and Pudles, 1977), but this was postulated to be From the Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706. Received April 3, 1978. Supported by a National Institutes of Health Postdoctoral Fellowship to R.E.V. and grants to W.W.C. from the National Institutesof Health (GM-18938) and the National Science Foundation (BMS-16134). A preliminary report on this work has been presented (Viola, 1978). 0006-2960/78/0417-4111$01 zyxwvut .OO/O October 3, 1978 The binding of metal-ATP is pH independent in this region, with the V/K profile for MgATP in the presence of citrate re- flecting the catalytic pK of 6.2. Since the pK of MgATP zy (5.3) is not seen in the V/KM~ATP profile, it appears that yeast hex- okinase can utilize both MgATP and MgHATP equally well. In the reverse reaction, deprotonation of a group with pK 6.8 caused loss of activity. The temperature independence of this pK, and of the pK seen in the V/KM~ATP profile in the forward direction in the presence of citrate, suggests the presence of a carboxylate group on the enzyme which must be deprotonated for yeast hexokinase to catalyze the transfer of the y-phosphate of ATP to sugars and protonated to catalyze the reverse reaction. This was confirmed by the effects of or- ganic solvent perturbations on the pK, indicating a catalytic group on the enzyme of the neutral acid type. a result of enzyme dissociation and conformationahchanges at the active site. Early modification studies (Fasella and Hammes, 1963) concluded that sulfhydryl groups had no role in the catalytic process; however, more recent work (Otieno et a]., 1977) has shown the presence of a single thiol group at the active site whose derivatization leads to the loss of activi- ty. X-ray crystallographic studies of the yeast hexokinase cpmplex with substrates at 2.7-A resolution (Steitz et al., 1977) concluded that protein side chains were interacting with the 3- and 4-hydroxyl groups of the sugar substrate. When the resolution was refined to 2.1-A (Anderson et al., 1978), there appeared to be an aspartyl residue interacting with the 4- and 6-hydroxyl groups. Relatively little use has been made of the diagnostic power of steady-state kinetics in determining the groups on the en- zyme responsible for substrate binding and catalysis. Some preliminary work on yeast hexokinase (Bohnensack and Hofmann, 1969), over a limited pH range, concluded that the group responsible for a decrease in activity in the V,,, profile with a pK = 6.8 was probably a histidyl residue. This conclu- sion was later disputed by the modification studies mentioned above. We have undertaken a more extensive kinetic study of the reaction catalyzed by yeast hexokinase and report evidence 0 1978 American Chemical Society 4111