Search

In the absence of a structure of the closed form of phosphoglycerate kinase we have modified by site directed mutagenesis several of the residues which, on the basis of the open form structure, are likely to be involved in substrate binding and catalysis. Here we report on the kinetic and anion activation properties of the yeast enzyme modified at positions 65, 121 and 168. In each case an arginine, thought to be involved in the binding of the sugar substrate's non-transferable phosphate group, has been replaced by lysine (same charge) and by methionine (no charge). Km values for 3-phosphoglycerate of all six mutant enzymes are only marginally higher than that of the wild-type enzyme. Removing the charge associated with two of the three arginine residues appears to influence (as judged by the measured Km's) the binding of ATP. Although binding affinity is not necessarily coupled to turnover the substitutions which have the greatest effect on the Km's do correlate with the reduction in enzymes maximum velocity. The one exception to this generalisation is the R65K mutant which, surprisingly, has a significantly higher kcat than the wild-type enzyme. In the open form structure of the pig muscle enzyme each of the three substituted arginines residues are seen to make two hydrogen bonds to the sugar substrate's non-transferable phosphate. From this it might be expected that anion activation would be similarly affected by the substitution of any one of these three residues. Although the interpretation of such effects are complicated by the fact that one of the mutants (R65M) unfolds at low salt concentrations, this appears not to be the case. Replacing Arg121 and Arg168 with methionine reduces the anion activation whereas a lysine in either of these two positions practically destroys the effect. With the substitutions at residue 65 the opposite is observed in that the lysine mutant shows anion activation whereas the methionine mutant does not.

The three-dimensional structure of fructose-1,6-bisphosphate aldolase from human muscle has been determined at 3.0 A resolution by X-ray crystallography. The active protein is a tetramer of 4 identical subunits each of which is composed of an eight-stranded alpha/beta-barrel structure. The lysine residue responsible for Schiff base formation with the substrate is located near the centre of the barrel in the middle of the sixth beta-strand. While the overall topology of the alpha/beta-barrel is very similar to those found in several other enzymes, the distribution of charged residues inside the core of the barrel seems distinct. The quaternary fold of human muscle aldolase uses interfacial regions also involved in the subunit association of other alpha/beta-barrel proteins found in glycolysis, but exploits these regions in a manner not seen previously.

Site-directed mutagenesis has been used to produce a mutant form of yeast phosphoglycerate kinase (PGK) in which the 'basic patch' residue His 62 has been replaced by a glutamine residue. Using 1H-NMR spectroscopy, it was found that 3-phosphoglycerate (3-PG) binding to the mutant protein induces the same conformational effects as for wild-type PGK, although the affinity was reduced by 2- to 3-fold. Kinetic studies show both Km for 3-PG and Vmax to be increased by approximately 2-fold relative to the wild-type enzyme. These data are consistent with the suggestion that His 62 assists in the binding of the substrate to the enzyme.