Location of the essential cysteine residue of jack bean urease.

Jack bean urease is inactivated by the modification of about one cysteine residue per subunit with N-ethylmaleimide (NEM) or other thiol titrant. The location of this cysteine residue was identified. After blocking the unessential thiol groups with NEM, the essential cysteine was labeled with N-(4-dimethylamino-3,5-dinitrophenyl)maleimide (DDPM). The DDPM-labeled protein was cleaved with cyanogen bromide and a DDPM-fragment was purified by gel filtration and ion exchange chromatography. Amino-terminal amino acid sequence of the DDPM-labeled fragment corresponded to that of a cyanogen bromide fragment of urease, VCHHLDREIPEDLAFAHSRIRKKTIAAEDVLNDIGAISIISSDSQAM. The second residue, Cys-592 in native urease, was labeled with DDPM. Fluoride ion, which competitively inhibits urease activity, protected urease from inactivation by NEM. The dissociation constant of fluoride ion obtained from the rate of inactivation with NEM was essentially identical to both the kinetically and spectrophotometrically determined dissociation constants. These suggest that the essential cysteine is at or near the active site.