The Role of an Essential Histidine Residue of Yeast Alcohol Dehydrogenase

Abstract

1. Inactivation of yeast alcohol dehydrogenase by diethyl pyrocarbonate indicates that one histidine residue per enzyme subunit is necessary for enzymic activity. The inactivated enzyme regains its activity over a period of days. . pyrazole, which are characteristic of native enzyme. 3.The rate constant for the reaction of enzyme with diethyl pyrocarbonate has been determined over the pH range 5.5-9. The histidine residue involved has approximately the same pK, as free histidine, but is 10-fold more reactive than free histidine.Alcohol dehydrogenases from yeast and horse liver each possess a histidine residue, five residues removed from a cysteine residue which, on alkylation with iodoacetate or iodoacetamide, results in the total loss of enzymic activity [l]. It is possible that a histidine residue may be important in catalysis by both of these alcohol dehydrogenases. Accordingly, we have studied the reaction of yeast alcohol dehydrogenase with diethyl pyrocarbonate under conditions where this reagent reacts with histidine residues [2]. Other pyridine-nucleotide-dependent dehydrogenases are known to be inactivated by diethyl pyrocarbonate, e.g. pig heart lactate dehydrogenase [3] and ox liver glutamate dehydrogenase [4]. This paper presents studies on the inactivation of yeast alcohol dehydrogenase by diethyl pyrocarbonate, and on some of the properties of the modified enzyme. The interaction of diethyl pyrocarbonate with horse liver alcohol dehydrogenase has also been examined. A preliminary report of some of the work has been published [5]. MATERIALS AND METHODS MaterialsAll solutions were prepared using glass-distilled water. The sodium phosphate and sodium pyrophos-

Chemical modifications of the Na+– H+ antiport in Escherichia coli membrane vesicles

“…where A / A o is the fraction of activity remaining at time t, Zo is the initial concentration of (Et0)2C203, k is the bimolecular rate constant for reaction of (Et0)2C203 with the Na' -H' antiporter and k' is the first-order rate constant for hydrolysis of the reagent measured according to [14]. The main body of Fig.…”

“…22Na' efflux was measured at pH 7.0 under conditions in which an artificial Ay of 128 mM was concomitantly created (see Table 1). The Aly-activated Na' efflux rates ( V ) were corrected for the energy-independent, (Et0)2C203-insensitive, basal Na' efflux rate (V,) and expressed as a percentage of residual activity as a function of the concentration of reagent least in part, from the spontaneous decomposition of the histidyl reagent in aqueous solution [14]. In order to account for this complicating effect, Gomi and Fujioka [I51 have shown that the data should be expressed as:…”

Estimation of rate dissociation constants involving ternary complexes in reactions catalysed by yeast alcohol dehydrogenase

“…The equilibrium measurements and stopped-flow studies reported here provide this information and also allow estimation of other rate and dissociation constants in the overall catalytic mechanism. The results are discussed in relation to the role of known ionizations at the active site of the enzyme (Dickenson & Dickinson, 1975c, 1977 and permit some comparison with horse liver alcohol dehydrogenase (Branden et al, 1975). Gibson & Milnes (1964).…”

Chemical modification of the two histidine and single cysteine residues in the channel-forming domain of colicin E1

“…The effective concentration of DEP was determined by reaction with 10 mM imidazole, pH 7.5 (Dickenson & Dickinson, 1975). The tryptic peptide in 0.5 M potassium phosphate buffer, pH 6.5, at concentrations of 0.1-0.5 mg/ml, was incubated at room temperature or 25~ with 0.05-0.15 mM DEP.…”