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

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Abstract
At pH 7.5, inactivation of NAD kinase by bromoacetyl pyridine occurs in three independent pseudo‐first‐order stages. Below pH 7.5, only two stages are distinguishable. The final stage alkylation leads to total inactivation, the earlier stages to the formation of an enzyme species with modified properties. Effects of pH on the time courses of inactivation suggest that the first stage may involve a variable number of sulphydryl groups. The final stage probably involves alkylation of a sulphydryl, with abolition of NAD binding. 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. Inhibition by L‐malate and by free ATP may occur by attachment of these effectors at separate modifier sites.