Plant Science 159 (2000) 149–158 Significance of sulfhydryl groups in the activity of urease from pigeonpea (Cajanus cajan L.) seeds Punit K. Srivastava, Arvind M. Kayastha * School of Biotechnology, Faculty of Science, Banaras Hindu Uniersity, Varanasi 221 005, India Received 18 February 2000; received in revised form 12 July 2000; accepted 13 July 2000 Abstract Titration of urease from pigeonpea (Cajanus cajan L.), a hexameric protein (mol. wt. 480 000; subunit mol. wt. 80 000), with 5,5-dithiobis-(2-nitrobenzoate) (DTNB) reveals the presence of 5.82 0.13 ‘accessible’ sulfhydryl 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 pH 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 biphasic, with half of the activity lost more rapidly than the remaining half. 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 addition 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. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Urease; Pigeonpea; Active site groups; Thiol inactivation; Fluoride protection; Half-site reactivity www.elsevier.com/locate/plantsci 1. Introduction Urease (urea amidohydrolase; 3.5.1.5), catalyzes the hydrolysis of urea to form ammonia and car- bon dioxide. High concentrations of ammonia arising from these reactions, as well as the accom- panying pH elevation, have important implica- tions in medicine and agriculture. Urease serves as a virulence factor in human and animal infections of the urinary and gastrointestinal tracts (reviewed in [1]), while high urease activity during soil nitro- gen fertilization with urea causes loss of ammonia by volatilization, inducing plant damage by am- monia toxicity and soil pH increase [2]. The en- zyme also plays a critical role in the nitrogen metabolism of many microorganism and plants [1,3]. Even before the medical and agricultural importance of urease was appreciated, it has been shown to be a historical enzyme. In 1926, Sumner crystallized the urease from jack bean seeds [4]. Nearly 50 years later, urease from jack bean was shown to possess nickel [5]. Although most recent bacterial urease related studies have focused on the structure, analysis of genes and enzyme, how- ever, interest in plant enzyme has continued [1]. The most extensively studied plant urease is the homohexameric protein from jack bean, which contains two-nickel ions per subunit [6]. However, bacterial urease from Klebsiella aerogenes is the best-characterized [1]. Recently 3-D structure of K. aerogenes urease was determined at 2.2 A  re- solution [7]. Both enzymes possess a cysteine residue (Cys 592 and Cys 319 in jack bean and K. aerogenes, respectively); however, the bacterial and * Corresponding author. Tel.: +91-542-368331; fax: +91-542- 368693/368174. E-mail address: kayastha@indiatimes.com (A.M. Kayastha). 0168-9452/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S0168-9452(00)00343-5