The Effect of Alkylation on Substrate‐ and Effector‐Binding Sites in Pigeon Liver NAD Kinase

Abstract

1. At pH 7.5, inactivation of NAD kinase by bromoacetyl pyridine occurs in three independent 2. Below pH 7.5, only two stages are distinguishable. 3. The final stage alkylation leads to total inactivation, the earlier stages to the formation 4. Effects of pH on the time courses of inactivation suggest that the first stage may involve 5. The final stage probably involves alkylation of a sulphydryl, with abolition of NAD binding. 6. No central role in catalysis can be postulated for any of the groups modified by bromoacetyl pyridine; two groups known to participate in catalysis and the binding of ATP * Mg are apparently not alkylated. 7.Inhibition by L-malate and by free ATP may occur by attachment of these effeotors at separate modifier sites.pseudo-first-order stages.of an enzyme species with modified properties. a variable number of sulphydryl groups.Steady-state kinetic studies of pigeon liver NAD kinase suggested a mechanism of action in which the free enzyme and binary enzyme-substrate complexes were in equilibrium with two or more ternary complexes, interconversion of which was the ratelimiting step [1,2]. The pH dependence of the parameters of the rate-equation also suggested the pKa values for a group concerned in the binding of ATP . Mg2-, and for another implicated in the catalytic step. Little other information as to the nature of the active site is available ; but it has been shown [3] that the alkylating agent 3-(bromoacetyl) pyridine inactivates the enzyme, first by the rapid modification of two or more groups to form a partially active species, then by the slow alkylation of a third residue, producing complete inactivation. The identity of these residues was not known, nor was their role in substrate binding, catalysis or the maintenance of enzyme configuration. In the work reported here, the effect of alkylation has been further investigated. Enzyme Irtactivators o-Iodosobenzoic acid and p-chloromercuribenzoic acid were obtained from Sigma: they were dissolved in dilute NaOH, neutralized, and used without delay. N-Ethylmaleimide (Sigma) was dissolved in water. 3-(Bromoacety1)-pyridine was prepared as the hydrobromide as described elsewhere [3]; it was dissolved in water and used immediately. Enzyme NAD kinase was purified from fresh pigeon liver homogenates, essentially as described previously [l]. Specific activity was 15-18 pmoles NADP+ formed x mg protein-l x h-l under the conditions described below. The preparation was free of malate dehydrogenase activity. MATERIALS AND METHODS Substrates and Inhibitors BuffersFor assays of NAD kinase over the pH range 5 .O -9.8, acetate, imidazole , triethanolamine , bicine and glycine buffers were employed. N ,N-bis-(2-hydroxyethyl)-glycine ("bicine") was obtained from British Drug Houses.It seemed likely that bromoacetyl pyridine would react with imidazole, and with the primary amines Tris and glycine; and anomalous results were obtained if these buffers were used during inactivation measurements. Maleate and borate were found to inhibit NAD kinase. For ti...

The Synthesis of Adenine‐Substituted Derivatives of NADP+ and Their Potential as Active Coenzymes and Affinity Adsorbents

“…amide] (6 pmol) was incubated with NAD' kinase (10 mg, 0.15 units) [21] and ATP (10 ymol) in 250 mM Tris-HC1 pH 7.5 (3 ml) for 1.5 days at 37°C. The reaction was followed by thin-layer chromatography and the appearance of activity with glucose-6-phosphate dehydrogenase.…”

“…Furthermore, on silica gel in solvent system B (Table 1) [21] in the presence of ATP under conditions where free NAD+ was quantitatively transformed into NADP' , as indexed by thin-layer chromatography and reduction with yeast glucose-6-phosphate dehydrogenase. In these studies enzyme was precipitated during the course of the incubation.…”