title: Site-directed mutagenesis of the ionizable groups in the active site of Zymomonas mobilis pyruvate decarboxylase: effect on activity and pH dependence.
abstract: Pyruvate decarboxylase (PDC, EC 4.1.1.1) is a thiamin diphosphate-dependent enzyme about which there is a large body of structural and functional information. The active site contains several absolutely conserved ionizable groups and all of these appear to be important, as judged by the fact that mutation diminishes or abolishes catalytic activity. Previously we have shown [Schenk, G., Leeper, F.J., England, R., Nixon, P.F. & Duggleby, R.G. (1997) Eur. J. Biochem. 248, 63-71] that the activity is pH-dependent due to changes in kcat/Km while kcat itself is unaffected by pH. The effect on kcat/Km is determined by a group with a pKa of 6.45; the identity of this group has not been determined, although H113 is a possible candidate. Here we mutate five crucial residues in the active site with ionizable side-chains (D27, E50, H113, H114 and E473) in turn, to residues that are nonionizable or should have a substantially altered pKa. Each protein was purified and characterized kinetically. Unexpectedly, the pH-dependence of kcat/Km is largely unaffected in all mutants, ruling out the possibility that any of these five residues is responsible for the observed pKa of 6.45. We conjecture that the kcat/Km profile reflects the protonation of an alcoholate anion intermediate of the catalytic cycle.
authors: Centre for Protein Structure, Function and Engineering, Department of Biochemistry and Molecular Biology, The University of Queensland, Brisbane, Australia.
authors: C Y
authors: CY
authors: Huang
authors: A K
authors: AK
authors: Chang
authors: P F
authors: PF
authors: Nixon
authors: R G
authors: RG
authors: Duggleby
mesh_terms: D001665:Binding Sites; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D011767:Pyruvate Decarboxylase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017355:Zymomonas / Q000201:enzymology*
chemical_list: D011767:Pyruvate Decarboxylase