Reactivity of the essential thiol of Klebsiella aerogenes urease. Effect of pH and ligands on thiol modification.
The kinetics of Klebsiella aerogenes urease inactivation by disulfide and alkylating agents was examined and found to follow pseudo-first-order kinetics. Reactivity of the essential thiol is affected by the presence of substrate and competitive inhibitors, consistent with a cysteine located proximal to the active site. In contrast to the results observed with other reagents, the rate of activity loss in the presence of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) saturated at high reagent concentrations, indicating that DTNB must first bind to urease before inactivation can occur. The pH dependence for the rate of urease inactivation by both disulfide and alkylating agents was consistent with an interaction between the thiol and a second ionizing group. The resulting macroscopic pKa values for the 2 residues are less than 5 and 12. Spectrophotometric studies at pH 7.75 demonstrated that 2,2'-dithiodipyridine (DTDP) modified 8.5 +/- 0.2 mol of thiol/mol of enzyme or 4.2 mol of thiol/mol of catalytic unit. With the slow tight binding competitive inhibitor phenyl-phosphorodiamidate (PPD) bound to urease, 1.1 +/- 0.1 mol of thiol/mol of catalytic unit were protected from modification. PPD-bound DTDP-modified urease could be reactivated by dialysis, consistent with the presence of one thiol per active site. Analogous studies at pH 6.1, using the competitive inhibitor phosphate, confirmed the presence of one protected thiol per catalytic unit. Under denaturing conditions, 25.5 +/- 0.3 mol of thiol/mol of enzyme (Mr = 211, 800) were modified by DTDP.
Todd
M J
MJ
Department of Biochemistry, Michigan State University, East Lansing 48824-1101.
Hausinger
R P
RP
D015082:2,2'-Dipyridyl / Q000031:analogs & derivatives* / Q000494:pharmacology; D004220:Disulfides / Q000494:pharmacology*; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D007460:Iodoacetamide / Q000494:pharmacology; D007700:Kinetics; D007709:Klebsiella / Q000201:enzymology*; D008024:Ligands; D013438:Sulfhydryl Compounds / Q000737:chemistry*; D014510:Urease / Q000037:antagonists & inhibitors / Q000737:chemistry*
D004220:Disulfides; D008024:Ligands; D013438:Sulfhydryl Compounds; C001105:2,2'-dipyridyl disulfide; D015082:2,2'-Dipyridyl; D004228:Dithionitrobenzoic Acid; D014510:Urease; D007460:Iodoacetamide