Use of site-directed mutagenesis and alternative substrates to assign the prototropic groups important to catalysis by Escherichia coli aspartate aminotransferase.
The pH dependence of Escherichia coli aspartate aminotransferase (AATase) has been investigated by the use of site-directed mutants and alternative substrates. Inhibition of the enzyme by CHES and variations in ionic strength are proposed to explain some of the qualitative differences in the published pH dependence of pig cytosolic AATase kinetics [Velick, S. F., & Vavra, J. (1962) J. Biol. Chem. 237, 2109-2122; Kiick, D.M., & Cook, P.F. (1983) Biochemistry 22, 375-382]. The pKa values of the basic limbs in the kcat/KM profiles for the amino acids, L-Asp and L-cysteinesulfinate (L-CS), are identical, within error, to those of free substrates, (L-Asp, pKa = 9.6; L-CS, pKa = 9.0). This pKa therefore is assigned to the alpha-amino group of the substrate. Replacement of the active site base, Lys-258, with the weaker base, gamma-thia-Lys, does not alter the intrinsic pKa for the profiles of the Ki values for the maleate-E.PMP complexes or the kcat/K alpha-KGM values. The mutation Y225F results in an alkaline shift of the pKa in the kcat/K alph-KGM profile. This pKa is assigned to the C4' amino group of PMP. E. coli AATase, unlike pig cytosolic AATase, shows a pH dependence on kcat between pH 5 and 10 that arises from a change in the rate-determining step at pH extremes. C alpha proton abstraction is partially rate-determining at neutral pH values, but not at pH extremes.(ABSTRACT TRUNCATED AT 250 WORDS)
Gloss
L M
LM
Department of Molecular and Cell Biology, University of California, Berkeley 94720.
Kirsch
J F
JF
D001219:Aspartate Aminotransferases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D002151:Calorimetry; D002384:Catalysis; D003545:Cysteine / Q000031:analogs & derivatives; D004926:Escherichia coli / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008239:Lysine; D008433:Mathematics; D016297:Mutagenesis, Site-Directed; D009994:Osmolar Concentration; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D013654:Taurine / Q000031:analogs & derivatives / Q000494:pharmacology
D011994:Recombinant Proteins; C050927:2-(N-cyclohexylamino)ethanesulfonic acid; D013654:Taurine; C000204:S-2-aminoethyl cysteine; D001219:Aspartate Aminotransferases; D008239:Lysine; D003545:Cysteine