Nucleotide binding to nucleoside diphosphate kinases: X-ray structure of human NDPK-A in complex with ADP and comparison to protein kinases

NDPK-A, product of the nm23-H1 gene, is one of the two major isoforms of human nucleoside diphosphate kinase. We analyzed the binding of its nucleotide substrates by fluorometric methods. The binding of nucleoside triphosphate (NTP) substrates was detected by following changes of the intrinsic fluorescence of the H118G/F60W variant, a mutant protein engineered for that purpose. Nucleoside diphosphate (NDP) substrate binding was measured by competition with a fluorescent derivative of ADP, following the fluorescence anisotropy of the derivative. We also determined an X-ray structure at 2.0A resolution of the variant NDPK-A in complex with ADP, Ca(2+) and inorganic phosphate, products of ATP hydrolysis. We compared the conformation of the bound nucleotide seen in this complex and the interactions it makes with the protein, with those of the nucleotide substrates, substrate analogues or inhibitors present in other NDP kinase structures. We also compared NDP kinase-bound nucleotides to ATP bound to protein kinases, and showed that the nucleoside monophosphate moieties have nearly identical conformations in spite of the very different protein environments. However, the beta and gamma-phosphate groups are differently positioned and oriented in the two types of kinases, and they bind metal ions with opposite chiralities. Thus, it should be possible to design nucleotide analogues that are good substrates of one type of kinase, and poor substrates or inhibitors of the other kind.     

Reverse and forward reactions of carbamyl phosphokinase from Streptococcus faecalis R. Participation of nucleotides and reaction mechanisms

The participation of Mg complex of nucleoside diphosphates and nucleoside triphosphates in the reverse and forward reactions catalyzed by purified carbamyl phosphokinase (ATP : carbamate phosphotransferase, EC 2.7.2.2) of Streptococcus faecalis R, ATCC-8043 were studied. The results of initial velocity studies of approx. 1 mM free Mg2+ concentration have indicated that in the reverse reaction MgdADP was as effective a substrate as MgADP. The phosphoryl group transfer from carbamyl phosphate to MgGDP, MgCDP and MgUDP was also observed at relatively higher concentrations of the enzyme and respective magnesium nucleoside diphosphate. In the forward direction MgdATP was found to be as efficient a phosphate donor as MgATP. On the other hand, Mg complexes of GTP, CTP and UTP were ineffective even at higher concentrations of the enzyme and respective magnesium nucleoside triphosphate. Product inhibition studies carried out at non-inhibitory level of approx. 1 mM free Mg2+ concentration have revealed that the enzyme has two distinct sites, one for nucleoside diphosphate or nucleoside triphosphate and the other for carbamyl phosphate or carbamate, and its reaction with the substrates is of the random type. Further tests of numerical values for kinetic constants have indicated that they are partially consistent with the Haldane relationship which is characteristic of rapid equilibrium and random mechanism.     

Metabolic Functions of Microbial Nucleoside Diphosphate Kinases

This article summarizes research from our laboratory on two aspects of the biochemistry ofnucleoside diphosphate kinase from Escherichia coli—first, its interactions with several T4bacteriophage-coded enzymes, as part of a multienzyme complex for deoxyribonucleosidetriphosphate biosynthesis. We identify some of the specific interactions and discuss whetherthe complex is linked physically or functionally with the T4 DNA replicationmachinery, orreplisome. Second, we discuss phenotypes of an E. coli mutant strain carrying a targeteddeletion of ndk, the structural gene for nucleoside diphosphate kinase. How do bacteria lackingthis essential housekeeping enzyme synthesize nucleoside triphosphates? In view of the specificinteractions of nucleoside diphosphate kinase with T4 enzymes of DNA metabolism, howdoes T4 multiply after infection of this host? Finally, the ndk disruption strain has highlybiased nucleoside triphosphate pools, including elevations of the CTP and dCTP pools of7- and 23-fold, respectively. Accompanied by these biased nucleotide pools is a strong mutatorphenotype. What is the biochemical basis for the pool abnormalities and what are the mutagenicmechanisms? We conclude with brief references to related work in other laboratories.     

Evidence for GTP-binding protein involvement in the tyrosine phosphorylation of the T cell receptor zeta chain.

The zeta subunit of the T cell receptor (TCR) is a prominent substrate for a TCR-activated tyrosine kinase. Tyrosine phosphorylation of the zeta subunit in response to antibody-mediated receptor cross-linking was synergized in permeabilized T cells by either of two non-hydrolyzable GTP analogues, guanosine 5'-[gamma-thio]triphosphate (GTP gamma S) or guanosine 5'-[beta, gamma-imido]triphosphate Gpp(NH)p. ATP analogues did not significantly affect antibody-induced tyrosine phosphorylation. Unlike the GTP analogues, the GDP analogue guanosine 5'-[beta-thio]diphosphate (GDP beta S) did not enhance phosphorylation of zeta. The effect induced by the GTP analogues required TCR occupancy and was independent of protein kinase C. Taken together these observations implicate a GTP-binding protein in the modulation of TCR-induced tyrosine phosphorylation.     

Activation of rat liver adenylate cyclase by guanosine 5''-[beta,gamma-imido]triphosphate and glucagon. Existence of reversibly and irreversibly activated states of the stimulatory GTP-binding protein.

The effects of guanosine 5'-[beta-thio]diphosphate (GDP[S]) on the kinetics of activation of rat liver membrane adenylate cyclase by guanosine 5'-[beta,gamma-imido]triphosphate (p[NH]ppG) were examined. GDP[S] caused immediate inhibition of the activation by p[NH]ppG at all time points tested. Substantial inhibition by GDP[S] was observed even after the time required for the enzyme to reach its steady-state activity, but the extent of inhibition became progressively smaller as the preincubation time with p[NH]ppG increased. The rate at which adenylate cyclase became quasi-irreversibly activated was a strictly first-order process. In the presence of glucagon, the formation of the irreversibly activated state was much slower. A combination of GDP[S] and glucagon could partially reverse the quasi-irreversible activation by p[NH]ppG. Glucagon decreased the lag time required for p[NH]ppG to activate adenylate cyclase and increased the extent of activation by p[NH]ppG. This stimulatory effect of the hormone on top of guanine nucleotide decreased on preincubation with p[NH]ppG, but not with GTP. Our results suggest that the activation of adenylate cyclase by non-hydrolysable GTP analogues is a two-stage process: the formation of a reversibly activated form (G rev) is a rapid process, followed by a much slower formation of the quasi-irreversibly activated form (G irr). Glucagon can stimulate G rev but not G irr, and can partially facilitate the formation of the G rev from the G irr state.