The Substrate Specificity of Pigeon Liver NAD Kinase

Abstract

1. The specificity of pigeon liver NAD kinase for both substrates and for divalent cations has been investigated. 2. The enzyme is activated by Mg++, Co++, Mn++, and Zn++ only: the ATPmetal complexes have different Km, values but the maximum velocities of the reactions are the same. 3. All nucleoside triphosphates investigated will act as substrates, although the Km values and maximum velocities differ. 4. The only NAD analogue found to act as a substrate was 3-acetyl pyridine-adenine dinucleotide (APAD). 5. The enzyme is inhibited by free Mg and free ATP. 6. The independence of substrate binding sites confirms that a random-addition, rapid equilibrium mechanism applies to NAD kinase.I n a previous paper [I] some kinetic work on NAD kinase (ATP : NAD 2'-phosphotransferase) was reported, and it was concluded that the enzyme acts by a mechanism in which the substrates bind rapidly, and in random order, to independent binding sites, the rate-limiting step of the reaction being interconversion of central ternary complexes. The case for such a mechanism would be strengthened if it could be shown that replacement of one substrate by an analogue with a different K , or Vmax had no effect on the binding of the other substrate: with this in mind, all three participants in the forward component of the reaction ATP * M= + +NAD= + ADP -M-+ +NADP4-+ H+ viz. the metal ion, the phosphate donor (nucleoside triphosphate) and the phosphate acceptor (pyridine nucleotide coenzyme) have been independently varied, and the kinetic parameters tabulated.

Kinetic Studies of the Interaction of Pigeon‐Liver NAD Kinase with Adenine Nucleotides and Divalent Cations

“…Previous kinetic results with NAD kinase [8,11,141 were consistent with a mechanism of action in which the substrates NADf and ATPMa-were bound to the enzyme rapidly and in random order, the ratelimiting step of the reaction being the interconversion of two or more ternary complexes. The rate equation for such a scheme, in Dalziel's notation [20], is :…”

“…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 concentrations of ATP4-and Ma+ present. Preliminary evidence was gained for inhibition of pigeon-liver NAD kinase by ATP [ 11,151 and by Mg2+ [ 111 ; such inhibition could be due to the formation of dead-end complexes with the enzyme, or of inactive or in-Enzyme NAD kinase was purified from high-speed supernatants of fresh isotonic pigeon liver homogenates, essentially as described previously [S], to a specific activity of 0.3 unitslmg protein. It was free of adenylate b a s e and NAD glycohydrolase activity, and contained less than 1 O l 0 ATPase activity.…”

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

“…In the E-NAD+ complex, k, has only 5 O/ , , of its value in the free enzyme, whilst in E-NADH it is zero ; thus the group attacked in the terminal process may be intimately concerned with NAD+ binding, or in the maintenance of the structure of the binding site ; and alkylation of this group might abolish binding of NAD+. ATP -Mn2-has a slighter effect on k, , but it could still be a direct one, since the two substrate sites, although independent [1,2], must obviously be adjacent for phosphate transfer to occur, particularly if, as seems likely, this occurs by direct nucleophilic attack by the 2'-hydroxyl of NAD+, on the y-phosphate of ATP. In creatine kinase the formerly non-ionizing sulphydryl becomes freely ionizing in the dead-end quaternary complex [S] ; the analogous situation with NAD kinase has not yet been investigated.…”

“…; i t j Fig.12). ATP4-has previously been shown to produce inhibition which is mixed with respect to both substrates [2]. The mode of inhibition by these substances was investigated by performing rate measurements with five independently varied concentrations of each substrate, in the presence of fixed concentrations of malate and free ATP, a t pH 7.3.…”

“…must have some inessential function, since their alkylation only partially abolishes activity, and under some circumstances (Table 2) actually enhances substrate binding. (With the random addition, rapid equilibrium mechanism proposed for NAD kinase [1,2], each K , should equal K g , the dissociation constant of a binary complex.) From its pH dependence, ko is likely to relate t o a sulphydryl group ; the approximately linear pH dependence of is difficult to explain, but it may be that the points in Fig.7, which show some scatter, could be fitted to a titration-type curve.…”