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. 2003 Nov;185(22):6732-5.
doi: 10.1128/JB.185.22.6732-6735.2003.

A mutation in the essential gene gmk (encoding guanlyate kinase) generates a requirement for adenine at low temperature in Salmonella enterica

Brian J Beck  1 , Mike Huelsmeyer, Scott Paul, D M Downs
Affiliations

A mutation in the essential gene gmk (encoding guanlyate kinase) generates a requirement for adenine at low temperature in Salmonella enterica

Brian J Beck et al. J Bacteriol. 2003 Nov.

Abstract

In Salmonella enterica serovar Typhimurium, gmk encodes guanylate kinase, an essential enzyme involved in the synthesis and salvage of guanine nucleotides. Here we report the isolation of a mutation in gmk that results in a nutritional requirement for adenine at low temperature. Comparisons of kinetic parameters from the wild-type and mutant Gmk enzymes revealed that the mutant enzyme had a more than 20-fold-higher Km for ATP than the wild-type enzyme. The growth dependence of the mutant on temperature and/or adenine could not be explained as a direct result of this kinetic difference. We propose a model in which previously described regulatory effects of GMP are responsible for these phenotypes.

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Figures

FIG. 1.
FIG. 1.
Simplified schematic representation of purine biosynthesis and salvage. The enzymes relevant to the processes discussed herein are identified by gene symbols below the reactions they catalyze; guanylate kinase (gmk) is shown in bold. Sites of allosteric inhibition by GMP that are significant for the model proposed are indicated. Abbreviations: A, adenine; G, guanine; Hx, hypoxanthine; HxR, inosine; AR, adenosine; GR, guanosine; apt, adenine phosphoribosyltransferase; gpt, guanine phosphoribosyltransferase; deoD, purine nucleoside phosphorylase; ndk, nucleoside diphosphate kinase. (While the DeoD-catalyzed reaction is reversible, the phosphorolysis of adenosine has been shown to be of minor quantitative importance.)
FIG. 2.
FIG. 2.
Kinetic analysis of Gmk and GmkS19N enzymes. Kinetic analyses were performed with wild-type (A) and mutant (B) Gmk enzymes. Initial velocity measurements were made using 5.1 nmol of enzyme in each reaction. The phosphoryl transfer of ATP to GMP was coupled to the pyruvate kinase-lactate dehydrogenase enzymes such that the absorbance change at 340 nm, detecting the decrease in NADH, revealed the guanylate kinase activity (8). In all cases the concentration of GMP was held constant at 3 mM and the concentration of ATP ranged from 0.015 mM to 10 mM. Kinetic analyses were carried out at both 37°C (▪) and 30°C (▴). The kinetic constants derived from these data are given in Table 2.
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