SUBSTRATE-SPECIFICITY OF HUMAN NUCLEOSIDE-DIPHOSPHATE KINASE REVEALEDBY TRANSIENT KINETIC-ANALYSIS

Nucleoside diphosphate kinases (NDKs) catalyze the transfer of gamma-p hosphoryl 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 r ates of phosphorylation and dephosphorylation with a range of nucleoti des and nucleotide analogues, For natural nucleotides, the rates of ph osphorylation and dephosphorylation were linearly dependent upon nucle otide concentration until they became tao fast to measure. The second order rate constants fori phosphorylation by natural NTPs varied betwe en 0.7 and 13 x 10(6) M-1 s(-1). Dephosphorylation by NDPs was 2-3-fol d faster than the corresponding phosphorylation reaction, and dephosph orylation by dNDPs was 3-4-fold slower than the equivalent NDPs. In al l cases, second order rate constants were highest for guanine followed by adenine and lowest for cytosine nucleotides. NDK also catalyzes th e transfer of thiophosphate from adenosine 5'-O-(thiotriphosphate) (AT P gamma S) and guanosine 5'O-(thiotriphosphate) (GTP gamma S) to NDP, but at 1/1000 of the equivalent phosphoryl transfer rates, hn this eas e, the observed rate constants of phosphorylation and dephosphorylatio n were hyperbolically dependent on nucleotide concentration, Thiophosp horylation ATP gamma S and GTP gamma S occurred with k(max) of 2.8 and 1.35 s(-1) and K-d of 145 and 36 mu M respectively, For dethiophospho rylation by a range of NDPs, k(max) was in the range of 5-30 s(-1), wh ereas K-d varied between 0.16 and 3.3 mM. Guanine had the lowest K-d v alues, and cytosine had the highest. The data are consistent with fast reversible binding of the nucleotide followed by the rate-limiting ph osphoryl transfer, Thiophosphates change only the rate of the phosphor yl transfer step, whereas both events are influenced by the base, Modi fication at the 2'-hydroxyl of ribose has only a small effect, while t he overall rate of phosphoryl transfer is reduced 1000-fold by modific ation at the 3'-ribose.