Substrate Specificity of Human Nucleoside-diphosphate Kinase Revealed by Transient Kinetic Analysis*

Nucleoside-diphosphate kinases (NDKs) catalyze the transfer of γ-phosphoryl groups from NTPs via an active site histidine to NDPs using a ping-pong mechanism. We have used the change of intrinsic tryptophan fluorescence that occurs upon phosphorylation of NDK to measure the rates of phosphorylation and dephosphorylation with a range of nucleotides and nucleotide analogues. For natural nucleotides, the rates of phosphorylation and dephosphorylation were linearly dependent upon nucleotide concentration until they became too fast to measure. The second order rate constants for phosphorylation by natural NTPs varied between 0.7 and 13 × 106 m −1s−1. Dephosphorylation by NDPs was 2–3-fold faster than the corresponding phosphorylation reaction, and dephosphorylation by dNDPs was 3–4-fold slower than the equivalent NDPs. In all cases, second order rate constants were highest for guanine followed by adenine and lowest for cytosine nucleotides. NDK also catalyzes the transfer of thiophosphate from adenosine 5′-O-(thiotriphosphate) (ATPγS) and guanosine 5′-O-(thiotriphosphate) (GTPγS) to NDP, but at 1 1000 of the equivalent phosphoryl transfer rates. In this case, the observed rate constants of phosphorylation and dephosphorylation were hyperbolically dependent on nucleotide concentration. Thiophosphorylation by ATPγS and GTPγS occurred withk max of 2.8 and 1.35 s−1 andK d of 145 and 36 μm respectively. For dethiophosphorylation by a range of NDPs, k maxwas in the range of 5–30 s−1, whereasK d varied between 0.16 and 3.3 mm. Guanine had the lowest K d values, and cytosine had the highest. The data are consistent with fast reversible binding of the nucleotide followed by the rate-limiting phosphoryl transfer. Thiophosphates change only the rate of the phosphoryl transfer step, whereas both events are influenced by the base. Modification at the 2′-hydroxyl of ribose has only a small effect, while the overall rate of phosphoryl transfer is reduced 1000-fold by modification at the 3′-ribose.

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