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 Uni6ersity, 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 9 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 9 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.comlocateplantsci
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- 368693368174.
E-mail address
:
kayasthaindiatimes.com A.M. Kayastha. 0168-945200 - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved.
PII: S0168-94520000343-5
plant enzyme thiols differ in their chemical proper- ties [8,9].
The urease active site is conserved among all sources as shown by extensive identity in the
protein sequences, a common requirement for nickel, and similarity in behavior towards in-
hibitors and inactivating reagents. For example, the presence of a thiol in jack bean urease is
supported by studies showing that alkylating and disulfide reagents inactivate the enzyme [8,10].
Similarly, disulfides inactivate K. aerogenes urease, apparently by disulfide exchange with the active
site cysteine [9,11]. It has been suggested that the slow loss of jack bean urease activity in the pres-
ence of b-mercaptoethanol and oxygen was due to the formation of mixed disulfide involving a
thiol located at the active site [12]. Titration of native jack bean urease has been carried out with
NEM [10,13] and 5,5-dithiobis-2-nitrobenzoate DTNB [10] and a pK
a
value of 9.15 at 25°C has been assigned to the unique cysteine [8]. The
amino acid sequence has been reported for a 34- residue cyanogen bromide peptide, which contains
this residue [14]. In contrast, urease from Staphy- lococcus xylosus has a threonine, instead of cys-
teine, at the active site; it is not inhibited by the SH group inhibitor and its DNA sequence encodes
no cysteine residue at this position [15]. Very recent structural studies on urease from Bacillus
pasteurii have shown that Cys
322
plays a significant role in the catalytic process, even though it is not
essential [16,17]. Such detailed structural informa- tion on both the binuclear Ni centre in the enzyme
active site and the mode of thiol inhibition is vital for structure-based rational design of urease re-
lated drugs [16]. Alkylating reagents specific for thiol groups, like iodoacetate IA, iodoacetamide
IAM, NEM and p-CMB, have been shown to inhibit several microbial ureases thus, the micro-
bial ureases possess a thiol group [1].
Urease from dehusked pigeonpea Cajanus cajan L. seeds has been purified to apparent homogene-
ity, partially characterized [18,19], and shown to be an important enzyme for analytical purposes
[20 – 23]. In continuation of our work on urease characterization from pigeonpea, the present pa-
per describes the significance of sulfhydryl groups in the activity of urease. Relative reactivities of the
functional groups -SH and site – site heterogene- ity within the urease hexamer molecule have also
been described.
2. Materials and methods
