Biochemical Society Transactions (1999) 27
22
EVALUATION OF A KINETIC APPROACH TO THE DETERMINATION OF
CATALYTIC SITE CONTENT IN ENZYMES AND ENZYME-LIKE
CATALYSTS
SANJIV SONKARIA,
BROCKLEHURST
SHERAZ
GUL,
MARINA
RESMlNl
and
KEITH
Laboratory of Structural and Mechanistic Enzymology, Dept. of Biochemistry, Queen
Mary and Westfield College, University of London, London El 4NS, UK.
Macromolecules that provide catalysis are typified by enzymes [ I ] but include also
catalytic antibodies [2] and synthetic polymers [3,4] where, in some cases, cavities of
defined geometry can be created [5,6]. Kinetic characterisation [7] requires knowledge of
the molarity of functional sites, [Ed, to permit calculation of values of k,., from
In some cases, site concentrations may be
experimentally determined values of Vm
determined by the use of stoichiometric reaction of the intact site with a substrate or sitespecific inhibitor to produce a product that is readily observable by a physico-chemical
technique [El. A potentially more general method involves a combination of steady state
kinetics with excess of substrate over catalyst to determine V,, and single turnover
kinetics with excess of catalyst over substrate to determine the limiting value ( k : c ) of
the observed first-order rate constant ( kobr ) corresponding to saturation of substrate by
catalyst. For the simple model ( I ) k t g = k,? = k, and [EJ = V-lk,.
kl
E+S
ES
E+P
(1)
Little use [3,4] has been made of this method particularly for enzyme active sites and the
extent to which it is model-specific has been explored only for polyclonal catalytic
antibodies [9]. We here report (i) that the method is valid for an obvious extension of the
simple model by inclusion of non-productive binding [lo] and (ii) that when applied to
the hydrolysis of Suc-Ala-Ala-Pro-Phe4NA catalysed by a-chymotrypsin (at 6.6"C
where K, is lowered) it provides a site concentration closely similar to to that obtained by
using the well-known active site titrant, N-fmns-cinnamoyl imidazole [ I I].
I , Sinnott, M.L. (ed) (1998) Comprehensive Biological Catalysis, Academic Press,
London
2. Resmini, M.. Vigna, R., Simms, C., Barber, N., Hagi-Pavli, E.P., Watts, A.B.. Verma,
C.. Gallacher. G.. & Brocklehurst, K. (1997) Biochem. J. 326,279-287
3. Klotz, I.M. (1987) in Enzyme Mechanisms (Page, I.M., & Williams, A. eds) pp. 1434. Royal Society of Chemistry, London
4. Hollfelder, F., Kirby, A.J. & Tawfik, D.S. (1997) J. Am. Chem. SOC.119,9578-9579
5 . Pike, V.W., Wharton, C.W., Brocklehurst, K. & Crook, E.M. (1978) Biochem. SOC.
Trans. 6, 269-271
6. Wulff, G. (1995) Angew. Chem. Int. Ed Engl 34,1812-1832
7. Gul, S., Sreedharan, S.K. & Brocklehurst, K. (1998) Enzyme Assays Essential data,
John Wiley & Sons Ltd, UK
8. Brocklehurst, K. (1996) in Enzymology Labfax (Engel, P. ed) pp. 59-75
9. Resmini, M., Gul, S., Sonkaria, S., Gallacher, G. & Brocklehurst, K. (1998)
Biochem. SOC.Trans. 26, S170
10. Brocklehurst, K., Crook, E.M. & Wharton, C W. (1968) FEBS Lett. 6,565-571
I I . Schonbaum, G.R., Zemer, B. & Bender, M.L. (1961) J. Biol. Chem. 236,2930-2935
23
-
Rho-GDI Rac interaction by NMR.
24
I. Barsukov, L.-Y. Lian, R. Badii, K.-H. Sze, G.C.K. Roberts
Biological NMR Centre, University of Leicester, PO BOX 138,
Medical Sciences Building, University Road, Leicester LEI 9HN
A small GTPase Rac of the Rho family controls NADPH oxidase
activity in phagocytes. The protein acts as a molecular switch
cycling between active GTP-bound and inactive GDP-bound form.
In the rest state Rac is bound to the small guanine nucleotide
exchange inhibitor Rho-GDI, which controls partitioning of Rac
between cytosol and membrane. Rho-GDI structure consists of a
flexible unstructured N-terminal domain of -60 residues and a Cterminal domain that has immunoglobulin fold. The presence of
both domains is essential for the Rac binding. We used NMR
method to study Rac - Rho-GDI interaction. The formation of
tight 1: I complex has been demonstrated. The binding is abolished
upon the flexible domain cleavage. Resonances of free Rho-GDI
have been assigned using ["C,15N]-labelledprotein. Chemical shift
changes of Rho-GDI signals upon Rac bir$ing have been partially
assigned using uniformly and selectively N-labelled protein. The
changes in the folded domain are mainly located on one surface of
the protein. In the flexible domain most of the resonances are
affected by the Rac binding. A small group of signals from
neighbouring residues of the flexible domain shows extreme line
broadening. The results suggest direct interaction of Rac with a
localised area on the folded domain and a short stretch in the
flexible domain of Rho-GDI. The regions of the flexible domain
not involved in the direct interaction remain relatively mobile. The
model of the complex is presented.
Investigation of electrostatic and hydrogen bonding interactions of
caricain and caricain D158N with time-dependent inhibitors.
SYEED HUSSAIN', KIMBERLEY K. ALLEN', IAN F. CONNERTON', NICOLA J.
CUMMINGS'. SHERAZ GUL', AKAVISH KHAN', MARK A.J. TAYLOR', EMRYS
W. THOMAS', CHANDRA VERMA' and KEITH BROCKLEHURST'
25
'Laboratory of Structural and Mechanistic Enzymology, Dept. of Biochemistry, Queen
Mary and Westfield College, University of London, London El 4NS, UK. 'Protein
Engineering Dept., Institute of Food Research, Reading Laboratory, Earley Gate,
Whiteknights Road, Reading RG6 2EF, UK. 'Dept. of Biological Sciences, University
of Salford, Salford M5 4JW, UK. 'Protein Structure Research Group, Dept. of
Chemistry, University ofYork, Heslington, York YO1 5DD, UK.
C. Damblon #, C. LY Lian #, RF'. Soto *, MH Villadnres
Frere * and GCK Roberts #
The reactivity characteristics of members of the cysteine proteinase superfamily,
notably papain (EC 3.4.22.2) are profoundly influenced by both specific binding
interactions and electrostatic effects [14]. In the present study the effects of
introducing a cationic ligand into the electrostatic net close to the catalytic site of
caricain (papaya proteinase 0;EC 3.4.22.30), a companion cysteine proteinase of
papain have been investigated, using o-aminoalkyl2-pyridyl disulfides as thiol-specific
reactivity probes (R). These compounds react on the stopped-flow timescale and at pH
6.6 exist as the monocations H,N'-[CH,].-S-S-2-Py.
The effectiveness of binding
(K. = [E][R]/[ER]) and of reaction within the ER complex (k,, the rate constant for
ER -+ products) depends on the spatial separation of the cationic -'NH, from the
electrophilic reaction centre (n = 2 - 3 & 4).
Computer modelling reveals variation in binding modes with variation in spatial
separation (n) involving both hydrogen bonding and electrostatic interactions with the
carboxylate side chain of Asp158, the electrostatic influence nearest to the
Cys25/His159 catalytic site ion-pair motif. Analogous experiments with the caricain
AsplS8Asn mutant demonstrates that in the absence of the carboxylate side chain the
effectiveness of binding decreases despite the potential for an intricate hydrogen
bonding network with the side chain carbonyl oxygen (>C=O) of Asn.
I.
2.
3.
4.
Plou, F.J., Kowlessur, D., Malthouse, J.P.G., Mellor, G.W, Hartshom, M.J.,
Pinitglang, S., Patel, H., Topham, C.M., Thomas, E.W., Verma, C, &
Brocklehurst, K. (1996) J. Mol. Biol. 257, 1088-1 1 1 1
Brocklehurst, K., ODriscoll, M., Kowlessur, D., Phillips, I.R., Templeton, W.,
Thomas, E.W., Topham, C.M. & Wharton, C.W. (1989) Biochem. J. 257,309-310
Gul, S., Dhillon, P.S., Verma, C., Thomas, E.W., & Brocklehurst, K. (1996)
Biochem. SOC.Trans. 24,4733
Gul, S., Clarke, A,, Field, B., Thomas, M.P., Willenbrock, F., Pinitglang, S.,
Verma, C., Thomas, E.W., & Brocklehurst, K. (1997) Biochem. SOC.Trans. 25,
91s
A37
STRUCTURAL STUDIES OF HISTIDINES IN ZINC
D-LACTAMASES AND THEIR INTERACTIONS WITH
UWlBITORS
*, M Galleni *, JM
# Lkcester Biological NhlR Centre, University of Leicester, LEI 9HN.
Leicester, UK.
* Centre d'hgenerie des Proteines, Labomtoire d'Enzymologie, Universite de
Liege, B4ooO Liege, Belgium.
The Mactam antibiotics are among the most useful antibacterial
chemotherapeutic agents, but their efficiency is continuously c h a l l q e d by the
emergence of resistant strains of pathogenic bacteria. The selective pressure
arising fiom the widespread use (and sometimes overuse) of &lactam antibiotics
has led to the development of resistance Production of klactamases which
inactivate &lactam antibiotics is the most efficient resistance mechanism.
5lactumaxs which require a zinc ion have received relatively little attention.
For many years, the only known zinc Rlactumax (2BL)was produced by an
m m o u s strain of Raciffus cereus (R. caws). More recently however,
enzymes of this class have been detected in pathogenic strains of Bacteroides
fmgilis, Aemmonos hydmphila (A. hydmphila). Pseudomonos aemginosa.
Smtia marcexens and Stenotrophomonas maltophilia. Since they are
resistant to inhibitors of the serine 5lactamases including clavulanic acid, the
spread of zinc 5lactamasemediated bacterial resistance to 5lactam antibiotics
(includmg carbepenem) is hghtening.
All the seven presently sequenced ZBLS exhibit sequence similarities, and it is
likely that they are homologous and share a common catalytic mechanism. They
appear to f i a w q u e family, distinct from the other metallo-peptidases (e.g.
thermolysin and carboxypeptidaseA)
With the exception of A. hydmphila, all the seven known ZBtS have four
histidine residues in the active site which are involved in metal binding. The
fourth histidine residue is replaced by an asparagine rn AH.This replacement
has been proposed to be the cause of the differences observed in the behaviour
of this enzyme.
We present here a study of the histidine residues in two ZBLr : A. hydmphila
and R. cereus by hetenmuclear NMR.The histidine imidazole can be selectively
observed in u n i f i e l y '% labelled proteins. The interactions of the imidazole
with metals cation and with ZRL inhibitors are discussed.