THE ROLE OF RESIDUES GLUTAMATE-50 AND PHENYLALANINE-496 IN ZYMOMONAS-MOBILIS PYRUVATE DECARBOXYLASE

Several enzymes require thiamine diphosphate (ThDP) as an essential co factor, and we have used one of these, pyruvate decarboxylase (PDC; EC 4.1.1.1) from Zymomonas mobilis, as a model for this group of enzymes . It is well suited for this purpose because of its stability, ease of purification, homotetrameric subunit structure and simple kinetic pro perties. Crystallographic analyses of three ThDP-dependent enzymes [Mu ller, Lindqvist, Furey, Schulz, Jordan and Schneider (1993) Structure 1, 95-103] have suggested that an invariant glutamate participates in catalysis. In order to evaluate the role of this residue, identified i n PDC from Zymomonas mobilis as Glu-50, it has been altered to glutami ne and aspartate by site-directed mutagenesis of the cloned gene. The mutant proteins were expressed in Escherichia coli. Here we demonstrat e that substitution with aspartate yields an enzyme with 3% of the act ivity of the wild-type, but with normal kinetics for pyruvate. Replace ment of Glu-50 with glutamine yields an enzyme with only 0.5% of the c atalytic activity of the wild-type enzyme. Each of these mutant enzyme s has a decreased affinity for both ThDP and Mg2+. It has been reporte d that the binding of cofactors to apoPDC quenches the intrinsic trypt ophan fluorescence [Diefenbach and Duggleby (1991) Biochem. J. 276, 43 9-445] and we have identified the residue responsible as Trp-487 [Dief enbach, Candy, Mattick and Duggleby (1992) FEBS Lett. 296, 95-98]. Alt hough this residue is some distance from the cofactor binding site, it lies in the dimer interface, and the proposal has been put forward [D yda, Furey, Swaminathan, Sax, Farrenkopf and Jordan (1993) Biochemistr y 32, 6165-6170] that alteration of ring stacking with Phe-496 of the adjacent subunit is the mechanism of fluorescence quenching when cofac tors bind. The closely related enzyme indolepyruvate decarboxylase (fr om Enterobacter cloacae) has a leucine residue at the position corresp onding to Phe-496 but shows fluorescence quenching properties that are similar to those of PDC. This suggests that the fluorescence quenchin g is due to some perturbation of the local environment of Trp-487 rath er than to a specific interaction with Phe-496. This latter hypothesis is supported by our data: mutation of this phenylalanine to leucine, isoleucine or histidine in PDC does not eliminate the fluorescence que nching upon addition of cofactors.