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The solvolyses of phosphorocreatine (creatine phosphate) and models for phosphorocreatine have been investigated and the results are applied to the mechanism of action of creatine kinase (EC 2.7.3.2). A metaphosphate intermediate appears to be involved.
The VirA-VirG two-component system regulates the 30-gene vir regulon in response to host-released chemical signals. VirA is a homodimeric membrane-spanning histidine protein kinase. Here, we show that mutations in two essential VirA residues, His-474 and Gly-657, can be complemented by the formation of mixed heterodimers, indicating that each subunit of a VirA dimer transphosphorylates the opposite subunit. VirA contains a receiver domain that inhibits kinase activity. We use the forced heterodimer system to show that the two receiver domains of a VirA dimer act independently and that each inhibits the phosphoacceptor subdomain of the opposite subunit. We also demonstrate that merodiploid strains co-expressing constitutive VirA mutants and wild-type VirA show levels of vir gene expression far lower than haploid strains expressing just the constitutive alleles. The fact that wild-type VirA can actively block vir gene expression in the absence of phenolic signals suggests that it might have a phospho-VirG phosphatase activity. The receiver domain of VirA is essential for this activity, whereas residues H474 and G657 of the kinase domain are not required. Merodiploid strains co-expressing a constitutive VirA allele and an allele that is kinase inactive but proficient in the inhibitory activity show strongly inducible vir gene expression, indicating that the inhibitory activity is modulated by environmental signals.
A study of the steady-state kinetics of the crystalline brewer's yeast (Saccharomyces carlsbergensis) nucleoside diphosphokinase, with the magnesium complexes of the adenine and thymidine nucleotides as reactants, has led to a postulated kinetic mechanism which proceeds through a substituted enzyme. This agrees with the earlier conclusions of Garces and Cleland [Biochemistry 1969; 8:633-640] who characterized a reaction between the magnesium complexes of the adenine and uridine nucleotides. An advantage of using thymidine nucleotides as reactants is that they permit accurate, rapid and continuous assays of the enzymatic activity in coupled-enzymatic tests. Through measurements of the initial velocities and product inhibition studies, the Michaelis constants, maximum velocities, and inhibition constants could be evaluated for the individual substrates. Competitive substrate inhibition was encountered at relatively high substrate concentrations, which also permitted an evaluation of their ability to act as 'dead-end' inhibitors. The Michaelis constants for the 3'-azido-3'-deoxythymidine (AzT) analogues were also evaluated and, although these values were only somewhat higher than those of their natural substrates, the Km's for the adenine nucleotides as paired substrates were lower and the Vmax's were drastically reduced. The pharmacological implications of these observations are touched upon and extrapolated to the cases where therapeutic doses of AzT may be employed.
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The effects of ribosomes and Mg-2plus on the binding of GDP and GTP to elongation factor 2 (EF-2) have been studied by an improved filter-binding assay. Both ribosomes and Mg-2plus strongly inhibit the binding of GDP but have apparently no effect on the GTP binding to the enzyme. An apparent stimulation by ribosomes of GTP binding to EF-2 is time-dependent and parallels a concomitant increase of the GDP concentration in the incubation mixture. Based on these results and evidence obtained by other investigators it is suggested that changes in the GTP:GDP ratio associated with the elongation and termination reactions of protein synthesis cause conformational changes of the respective factors which consequently will modulate the binding and dissociation of the enzymes from ribosomes. Further evidence of the role GDP may play as a modulator of protein synthesis might possibly be provided by studies of the GTP-GDP transphosphorylase activity which is present as an impurity in highly purified preparations of EF-2 as well as in ribosome preparations. It is demonstrated that relatively high concentrations of GDP in the presence of GTP completely block the ribosome-dependent GTPase activity of EF-2. Instead, the transphosphorylase enzyme(s) catalyzes an exchange reaction between GTP and GDP during which GDP remains bound to EF-2 and the relative concentrations of the two nucleotides do not change.
Adenylate kinase (ATP:AMP transphosphorylase) is a key enzyme in energy metabolism. The activity of its isoforms is subjected to multiple regulations. It is shown here that a specific fraction consisting of all adenylate kinase isoforms from tobacco leaves and tissue cultures does not bind to the anionic exchange-resin Mono Q. Sample pretreatment with ribonuclease could restore full binding to Mono Q, suggesting an association of adenylate kinase with RNA similar to the enzyme of Chenopodium rubrum (J. Chromatogr. 625: 13-19). We propose here that at least in vitro adenylate kinase can behave as an RNA-binding protein and that RNA-binding of adenylate kinase isoforms may be related to regulatory mechanisms.