Site-directed mutagenesis of proline 204 in the 'hinge' region of yeast phosphoglycerate kinase

Site-specific mutants have been produced in order to investigate the role o f proline 204 in the 'hinge' region of yeast phosphoglycerate kinase (PGK). This totally conserved proline has been shown to be the only cis-proline i n the high resolution crystal structures of yeast, B. stearothermophilus, T . brucei and T. maritima PGK, and may therefore have a role in the independ ent folding of the two domains or in the 'hinge' bending of the molecule du ring catalysis. The residue was replaced by a histidine (Pro204His) and a p henylalanine (Pro204Phe), and the resulting proteins characterised by diffe rential scanning calorimetry (DSC), circular dichroism (CD), tryptophan flu orescence emission and kinetic analysis. Although the secondary and tertiary structure of the Pro204His protein is g enerally similar to that of the wild-type enzyme as assessed by CD, the enz yme is less stable to heat and guanidinium chloride denaturation than the w ild-type. In the denaturation experiments two transitions were observed for both the wild-type and the Pro204His mutant, as have been previously repor ted for yeast PGK [Missiakas, D., Betton, J.M., Minard, P. & Yen, J.M. (199 0) Biochemistry 29, 8683-8689]. The first transition is accompanied by an i ncrease in fluorescence intensity leading to a hyperfluorescent state, foll owed by the second, corresponding to a decrease in fluorescence intensity. However, for the Pro204His mutant, the first transition proceeded at lower concentrations of guanidinium chloride and the second transition proceeded to the same extent as for the wild-type protein, suggesting that sequence-d istant interactions are more rapidly disrupted in this mutant enzyme than i n the wild-type enzyme, while sequence-local interactions are disrupted in a similar way. The Michaelis constants (K-m) for both 3-phospho-D-glycerate and ATP are increased only by three or fourfold, which confirms that, as e xpected, the substrate binding sites are largely unaffected by the mutation . However, the turnover and efficiency of the Pro204His mutant is severely impaired, indicating that the mechanism of 'hinge' bending is hindered. The Pro204Phe enzyme was shown to be significantly less well folded than the w ild-type and Pro204His enzymes, with considerable loss of both secondary an d tertiary structure. It is proposed that the proline residue at 204 in the 'hinge' region of PGK plays a role in the stability and catalytic mechanis m of the enzyme.