1. NAD kinase is inhibited by free Mg2+, free ATP and by a number of other adenine nucleo-2. Inhibition by ATP, ADPMg-, ADP-ribose2-and AMP2-is competitive with respect to 3. ATP appears to bind equally well to the free enzyme and to binary enzyme substrate 4. The other adenine nucleotides studied have markedly less affinity for the free enzyme than 5. Mg2+ probably inhibits by forming a complex with NADt, which binds to the enzyme but 6. Low concentrations of Mg2+ reduce KgADf in an unexplained manner.7. ATPCa2-is a substrate for NAD kinase, but the rate-limiting step of the reaction may be tides.both ATPMg2-and NAD+.complexes.for binary complexes.is inactive in the reaction. different when other metals activate the enzyme.Although the ATP-dependent phosphotransferases have been widely studied in recent years, the role of divalent cations, which are obligatory cofactors for the reactions catalysed by these enzymes, is not completely understood. I n several instances [I -41 they are essential for the catalytic function of the enzyme but have little or no effect on the binding of adenine nucleotides, whereas in others [5] the metal ion may form a bridge between the protein and the nucleotide. Steady-state kinetic analysis can provide an indirect means of studying the interaction of such enzymes with the free nucleotide ATP4-or metal ion M2+, as well as the I : 1 complex, ATPM2-, which is the substrate of the reaction. I n the particular case of NAD kinase, previous studies [6-141 have usually been made with a small excess of divalent cation over ATP, so that the complex ATPM2-was the dominant species, with relatively low con- However, ATP afforded NAD kinase some protection from alkylation by bromoacetyl pyridine [17], and it was suggested that the free nucleotide might bind to the enzyme, perhaps a t a site separate from that occupied by ATPM2-. I n the present paper we report more detailed kinetic studies of the interaction of pigeon liver NAD kinase with ATP, Mg2+, Ca2+ and some other adenine nucleotides. MATERIALS AND METHODSReagents ATP (highest grade, from equine muscle), ADP and AMP were from Sigma. NAD+ was from Boehringer Mannheim GmbH (Mannheim, Germany). ADPribose was prepared by cleavage of NAD+ with Neurospora NAD glycohydrolase [ 181. Supplementary enzymes and their substrates were from Boehringer Mannheim GmbH (Mannheim, Germany).ATP, ADP and NAD+ were assayed as described previously [8] ; AMP and ADP-ribose were estimated by absorbance measurements at 260 nm. Ca2+, Co2+ and Mg2+ were used as their chlorides and Mn2+ and Zn2+ as their sulphates. CaCI, was standardized by titration against silver nitrate.

The reaction catalysed by creatine kinase was studied in both directions by quenched-flow techniques to follow the initial product formation in the millisecond range. In both directions the amount of product formed increases linearly with time, and the turnover number corresponds to the steady-state value. Extrapolation to zero time indicates the absence of either a large transient phase or a large lag phase in both directions. This indicates that the actual chemical reaction is rate-limiting, and that all possible isomerizations before or after the chemical step must be much more rapid.