Amino Acid Sequence around a Cysteine Residue in the Active Center of Jack Bean Urease

Abstract

Cysteine residues in the active center of jack bean urease [EC 3. 5. 1. 5] were modified with ¹⁴C-labeled diazonium-1 H-tetrazole (DHT). The labeled enzyme was carboxymethylated with iodoacetic acid, and then hydrolyzed with trypsin. The tryptic digest was subjected to gel filtration on Sephadex G-50, yielding two radioactive fractions. The [¹⁴C] DHT-labeled peptide having a lower molecular weight, which was determined to be approximately 1, 000 by the method of gel filtration, was further purified to homogeneity by ion-exchange chromatography on DEAE-Sephadex A-25. [¹⁴C] DHT-labeled cysteine was identified as cysteic acid after performic acid oxidation, and the amino acid sequence of the low-molecular-weight [¹⁴C] DHT-labeled peptide was determined to be Phe-Glu-Pro-Gly-Asp-Cys-Asn-Ser-Thr-Phe-Lys.