ИСТИНА

Glyceraldehyde-3-phosphate dehydrogenase is a glycolytic enzyme catalyzing oxidation and phosphorylation of glyceraldehyde-3-phosphate coupled with the reduction of NAD+. In humans, it is present in two homologous isoenzyme forms: somatic (GAPD) and spermatogenic (GAPDS). The recombinant GAPDS was previously discovered to exhibit enhanced stability towards denaturation as compared to GAPD. In the present study, GAPDS structural features possibly involved in its stabilization are examined. The sequences and x-ray structures of both isoenzymes were first analyzed in order to reveal any specific differences that might be concerned with stabilization. The GAPDS backbone was then discovered to be more rigid than the GAPD one because of higher proline and lower glycine contents. GAPDS was also found to contain some extra salt bridges, mostly interdomain or intersubunit. The site-directed mutagenesis approach was then applied to introduce six point mutations to the GAPDS sequence which were assumed to lower its stability. Three of them affected the extra proline residues (P111A, P157A and P326A), while the others disrupted some of the additional salt bridges (E96Q, E244Q and D311N). The subsequent experimental assessment of the mutant proteins revealed that the whole GAPDS molecule is destabilized by the P326A, E96Q and D311N substitutions. On the contrary, the P111A substitution had a surprisingly strong stabilizing effect. The active center was destabilized by merely the P326A and D311N substitutions and stabilized by the P111A substitution. Taken together, the evidence for GAPDS being significantly stabilized by the extra proline residue P326, as well as the additional interdomain salt bridges E96-H394 and D311-H124 was shown. These findings can be implicated for engineering the proteins with altered stability.