General base catalysis in the urate oxidase reaction: evidence for a novel Thr-Lys catalytic diad.
Urate oxidase catalyzes the oxidation of urate without the involvement of any cofactors. The gene encoding urate oxidase from Bacillus subtilis has been cloned and expressed, and the enzyme was purified and characterized. Formation of the urate dianion is believed to be a key step in the oxidative reaction. Rapid-mixing chemical quench studies provide evidence that the dianion is indeed an intermediate; at 15 degrees C the dianion forms within the mixing time of the rapid-quench instrument, and it disappears with a rate constant of 8 s(-)(1). Steady-state kinetic studies indicate that an ionizable group on the enzyme with a pK of 6.4 must be unprotonated for catalysis, and it is presumed that the role of this group is to abstract a proton from the substrate. Surprisingly, examination of the active site provided by the previously reported crystal structure does not reveal any obvious candidates to act as the general base. However, Thr 69 is hydrogen-bonded to the ligand at the active site, and Lys 9, which does not contact the ligand, is hydrogen-bonded to Thr 69. The T69A mutant enzyme has a V(max) that is 3% of wild type, and the K9M mutant enzyme has a V(max) that is 0.4% of wild type. The ionization at pH 6.4 that is observed with wild-type enzyme is absent in both of these mutants. It is proposed that these residues form a catalytic diad in which K9 deprotonates T69 to allow it to abstract the proton from the N9 position of the substrate to generate the dianion.
Imhoff
Rebecca D
RD
Department of Biochemistry, University of Missouri, Columbia, Missouri 65211, USA.
Power
Nicholas P
NP
Borrok
M Jack
MJ
Tipton
Peter A
PA
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D007700:Kinetics; D008239:Lysine / Q000378:metabolism*; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D013912:Threonine / Q000378:metabolism*; D014503:Urate Oxidase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D013912:Threonine; D014503:Urate Oxidase; D008239:Lysine