HIGHLY CONSERVATIVE SEQUENCE IN THE CARBOXYL-TERMINUS OF SARCOSINE OXIDASE IS IMPORTANT FOR SUBSTRATE-BINDING

The function of the highly conservative sequence --G(344)-F-S-G-H-G-F- K-F(352)-- in the carboxyl terminus of sarcosine oxidase was investiga ted using site-directed mutagenesis. When H-348 was substituted with u ncharged amino acids, the K-m values of sarcosine oxidase markedly inc reased, although the k(cat) values remained the same as that of the wi ld-type enzyme. The kinetic parameters obtained with other mutations a lso suggested that the conservative sequence acts as the substrate-bin ding site. When K-351 was replaced by Ala, the mutant K351A could not bind the coenzyme FAD (flavin adenine dinucleotide). This result sugge sted that K-351 interacts with the FAD-binding site of the amino-termi nal region. The enzymic activities of the mutants H348Q and H348A were lost at neutral and acidic pH as a result of the disappearance of the positive charge on H-348.