Significance of sulfhydryl groups in the activity of urease from pigeonpea(Cajanus cajan L.) seeds

Titration of urease from pigeonpea (Cajanus cajan L.), a hexameric protein (mol. wt. 480 000; subunit mel. wt. 80 000), with 5,5'-dithiobis-(2-nitrobe nzoate) (DTNB) reveals the presence of 5.82 +/- 0.13 'accessible' sulfhydry l groups per molecule of the enzyme protein (i.e. about one 'accessible' SH group per subunit). Denatured enzyme was found to titrate for 12.1 +/- 0.1 SH groups per molecule (i.e, about two SH groups per subunit). Half of the 'accessible' groups react faster than the remaining at pH 8.5 as well as p H 7.5. However, the reaction was slower at pH 7.5 than 8.5. Time-dependent loss of enzyme activity with DTNB was also found to be biphasic. The enzyme was inactivated at low concentration of p-chloromercuribenzoate (p-CMB), N -ethyl maleimide (NEM) and iodoacetamide. The inactivation reactions were b iphasic, with half of the activity lost more rapidly than the remaining hal f. The loss of activity with p-CMB was linearly related to the blocking of accessible SH groups. Inactivation by p-CMB is largely reversible by additi on of excess of cysteine. Fluoride ion strongly protects the enzyme against NEM inactivation, however, substrate urea provides much weaker protection against SH group reagents. The significance of these results is discussed. (C) 2000 Elsevier Science Ireland Ltd. All rights reserved.