Catalytic properties of enzymes used in biotechnology can be improved by eliminating those regulatory mechanisms that are not absolutely required for their functioning. We exploited mammalian glyceraldehyde-3-phosphate dehydrogenase as a model protein and examined the structural basis of the NAD(+) cooperative binding exhibited by its homologous isoenzymes: the somatic enzyme (GAPD) and the recombinant sperm-specific enzyme (dN-GAPDS). Moreover, we obtained a mutant dN-GAPDS, which misses the cooperativity, but exhibits a twofold increase in the specific activity instead (92 and 45 μmol NADH/min per mg protein for the mutant and the wild type proteins, respectively). Such an effect was caused by the disruption of the interdomain salt bridge D311-H124, which is located close to the active site of the enzyme. The thermal stability of the mutant protein also increased compared to the wild type form (heat absorption peak values were 70.4 and 68.6 °C, respectively). We expect our findings to be of importance for the purposes of biotechnological applications.
Keywords: Cooperativity; Glyceraldehyde-3-phosphate dehydrogenase; Ligand binding; Salt bridge.
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