REPLACEMENT OF ACTIVE-SITE LYSINE-239 OF THERMOSTABLE ASPARTATE-AMINOTRANSFERASE BY S-(2-AMINOETHYL)CYSTEINE - PROPERTIES OF THE MUTANT ENZYME

The active-site lysine residue of thermostable aspartate aminotransferase, Lys-239, to which the cofactor, pyridoxal 5'-phosphate (PLP), is bound, has been converted to Cys by site-directed mutagenesis. The thiol group of Cys-239 was chemically aminoethylated with ethylenimine. Amino acid analysis of the modified enzyme showed that it contained about 1 mol of S-(2-aminoethyl)cysteine (SAEC) per mol subunit. The activity of the mutant enzyme (K239SAEC) was about 14% of that of the wild-type enzyme. No significant difference in thermostability was found between the wild-type and K239SAEC enzymes. The UV-visible spectrum of K239SAEC showed a peak (lambda(max) 380 nm), due to absorption by the cofactor, at a 20 nm longer wavelength than that of the wild-type enzyme. The circular dichroism band due to the bound cofactor of K239SAEC also shifted toward a 20 nm longer wavelength. We determined kinetic parameters (rate constants, k(max), and dissociation constants, K(d), for the substrates) for each half transamination catalyzed by the wild-type and K239SAEC mutant enzymes by the stopped-flow method. The k(max) values for the mutant enzyme reactions were 2.6-24 times lower than those for the wild-type enzyme ones. The two enzymes showed similar K(d) values for the same substrates except glutamate; the mutant enzyme showed higher affinity for glutamate than the wild-type enzyme.