Initial velocity, spectral, and pH studies of the serine-glyoxylate aminotransferase from Hyphomicrobiuim methylovorum.
Serine-glyoxylate aminotransferase (SGAT) from Hyphomicrobium methylovorum is a pyridoxal 5'-phosphate (PLP) enzyme that catalyzes the interconversion of L-serine and glyoxylate to hydroxypyruvate and glycine. The initial velocity and dead-end inhibition patterns are consistent with a ping-pong kinetic mechanism. The Km values for L-serine and the alternative substrate ketomalonate are 0.28 +/- 0.02 and 1.13 +/- 0.08 mM, respectively. The spectrum of SGAT at pH 7.5 shows an absorbance maximum at 413 nm and a shoulder centered at 330 nm corresponding to the ketoenamine and enolimine forms of the protonated Schiff's base with the enolimine tautomer predominating. As determined by the changes in the enzyme absorbance spectrum the enzyme can be converted from the E-PLP to the E-pyridoxamine 5'-phosphate (E-PMP) form on addition of L-serine. The enzyme can subsequently be converted back to E-PLP by addition of glyoxylate or hydroxypyruvate. The enzyme displays a pH-dependent spectral change with a pK of about 8.2 which is ascribed to the ionization of an enzymatic residue that effects the tautomeric equilibrium between the ketoenamine and enolimine tautomers of the protonated aldimine. The V/K(L-serine) pH profile displays two pK values at pH 7.5 and 8.5 with limiting slopes of 1 and -1. The V/K(ketomalonate) pH profile displays one pK at 8.2 on the basic side with a limiting slope of 1 and the log K(I oxalate) pH profile shows one pK on the basic side at pH 7.2. The data suggest the active enzyme is the protonated aldimine and an enzymatic base with a pK of 7.5 accepts a proton from the alpha-amine of substrate to initiate catalysis.
Karsten
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WE
Department of Chemistry and Biochemistry, University of Oklahoma, Norman 73019, USA.
Ohshiro
T
T
Izumi
Y
Y
Cook
P F
PF
D006863:Hydrogen-Ion Concentration; D020582:Hyphomicrobium / Q000201:enzymology*; D007700:Kinetics; D013056:Spectrophotometry, Ultraviolet; D000637:Transaminases / Q000737:chemistry* / Q000378:metabolism
D000637:Transaminases; C022636:serine-glyoxylate aminotransferase