Contribution to catalysis and stability of the five cysteines in Escherichia coli aspartate aminotransferase. Preparation and properties of a cysteine-free enzyme.
The five cysteines, at positions 82, 191, 192, 270, and 401, of Escherichia coli aspartate aminotransferase (AATase) were, individually and in some combinations, converted to alanine by site-directed mutagenesis (C82A, C191A, C192A, C270A, C401A). Cys-191, which is conserved in all AATase isozymes, was mutated to serine as well (C191S). A quintuple mutant, with all cysteines converted to alanines (Quint), was also constructed. The effects of these single and multiple mutations were examined by steady-state kinetics and urea denaturation. The thermal stabilities of Quint and of the wild-type enzyme (WT) were determined by differential scanning calorimetry. The mutants had kcat values up to 50% greater than that of WT and KMAsp and KM alpha-KG values up to 1.5- and 3.3-fold higher than that of WT. The mutants C82A and C191A exhibit nearly the same CM in urea denaturation experiments as WT, while the other single mutants and Quint are less stable, with CM differences of up to 0.7 M urea. Quint is also less thermostable than WT, with a delta TM of 3.3-4.4 degrees C. Thus the five cysteine replacements yield small, but significant, changes in catalytic and denaturation parameters, but none of the cysteines was found to be essential. The changes manifested in the mutation of the conserved Cys-191 to alanine are no greater than those observed with the four nonconserved cysteines. We consider the evolutionary implications of these findings.
Gloss
L M
LM
Department of Molecular and Cell Biology, University of California, Berkeley 94720.
Planas
A
A
Kirsch
J F
JF
D000595:Amino Acid Sequence; D000818:Animals; D001219:Aspartate Aminotransferases / Q000737:chemistry* / Q000235:genetics; D002152:Calorimetry, Differential Scanning; D002384:Catalysis; D002645:Chickens; D003545:Cysteine / Q000737:chemistry*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology*; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011489:Protein Denaturation; D051381:Rats; D013053:Spectrophotometry; D013438:Sulfhydryl Compounds / Q000378:metabolism; D013552:Swine; D013816:Thermodynamics; D014422:Turkeys; D014508:Urea
D007527:Isoenzymes; D013438:Sulfhydryl Compounds; D014508:Urea; D001219:Aspartate Aminotransferases; D003545:Cysteine