Changes in the coordination geometry of the active-site metal during catalysis of benzylpenicillin hydrolysis by Bacillus cereus .beta.-lactamase II

Biochemistry ◽  
1986 ◽  
Vol 25 (22) ◽  
pp. 7208-7215 ◽  
Author(s):  
Roy Bicknell ◽  
Andreas Schaffer ◽  
Stephen G. Waley ◽  
David S. Auld
Keyword(s):  
Bacillus Cereus ◽  
Active Site ◽  
Coordination Geometry ◽  
Beta Lactamase ◽  
Beta Lactamase Ii

scholarly journals Site-directed mutagenesis of dicarboxylic acids near the active site of Bacillus cereus 5/B/6 β-lactamase II

1991 ◽  
Vol 276 (2) ◽  
pp. 401-404 ◽  
Author(s):  
H M Lim ◽  
R K Iyer ◽  
J J Pène
Keyword(s):  
Bacillus Cereus ◽  
Aspartic Acid ◽  
Carboxy Group ◽  
Active Site ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Beta Lactamase ◽  
Beta Lactam ◽  
Aspartic Acid Residue ◽  
Beta Lactamase Ii

An amino acid residue functioning as a general base has been proposed to assist in the hydrolysis of beta-lactam antibiotics by the zinc-containing Bacillus cereus beta-lactamase II [Bicknell & Waley (1985) Biochemistry 24, 6876-6887]. Oligonucleotide-directed mutagenesis of cloned Bacillus cereus 5/B/6 beta-lactamase II was used in an ‘in vivo’ study to investigate the role of carboxy-group-containing amino acids near the active site of the enzyme. Substitution of asparagine for the wild-type aspartic acid residue at position 81 resulted in fully functional enzyme. An aspartic acid residue at position 90 is essential for beta-lactamase II to confer any detectable ampicillin and cephalosporin C resistance to Escherichia coli. Conversion of Asp90 into Asn90 or Glu90 lead to the synthesis of inactive enzyme, suggesting that the spatial position of the beta-carboxy group of Asp90 is critical for enzyme function.

scholarly journals A thiono-β-lactam substrate for the β-lactamase II of Bacillus cereus. Evidence for direct interaction between the essential metal ion and substrate

1989 ◽  
Vol 258 (3) ◽  
pp. 765-768 ◽  
Author(s):  
B P Murphy ◽  
R F Pratt
Keyword(s):  
Bacillus Cereus ◽  
Active Site ◽  
Metal Ion ◽  
Direct Interaction ◽  
Beta Lactamase ◽  
Beta Lactam ◽  
Enzyme Active Site ◽  
Lactam Carbonyl ◽  
Beta Lactamase Ii ◽  
Hydrolysis Of

An 8-thionocephalosporin was shown to be a substrate of the beta-lactamase II of Bacillus cereus, a zinc metalloenzyme. Although it is a poorer substrate, as judged by the Kcat./Km parameter, than the corresponding 8-oxocephalosporin, the discrimination against sulphur decreased when the bivalent metal ion in the enzyme active site was varied in the order Mn2+ (the manganese enzyme catalysed the hydrolysis of the oxo compound but not that of the thiono compound), Zn2+, Co2+ and Cd2+. This result is taken as evidence for kinetically significant direct contact between the active-site metal ion of beta-lactamase II and the beta-lactam carbonyl heteroatom. No evidence was obtained, however, for accumulation of an intermediate with such co-ordination present.

scholarly journals β-lactamase I from Bacillus cereus. Structure and site-directed mutagenesis

1987 ◽  
Vol 248 (3) ◽  
pp. 657-662 ◽  
Author(s):  
P J Madgwick ◽  
S G Waley
Keyword(s):  
Bacillus Cereus ◽  
Active Site ◽  
Active Enzyme ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Beta Lactamase ◽  
Ampicillin Resistance ◽  
Mutant Genes ◽  
Conserved Residue ◽  
Invariant Residue

The sequence of the gene for beta-lactamase I from Bacillus cereus 569/H has been redetermined. Oligonucleotide-directed mutagenesis has been carried out, and the effects of the changes on the ampicillin-resistance of Escherichia coli TG1 expressing the mutant genes have been studied. Lysine-73, close to the active-site serine-70 and a highly-conserved residue, has been converted into arginine. This change had a large effect on activity, but did not abolish it. An even larger effect was found in the mutant in which glutamate-166 had been converted into glutamine; this had little or no activity. On the other hand, the conversion of glutamate-168 into aspartate gave fully active enzyme. Glutamate-166 is an invariant residue, but glutamate-168 is not. Alanine-123 has been replaced by cysteine, to give active enzyme; this change forms part of the plan to introduce a disulphide bond into the enzyme.

scholarly journals Identification of histidine residues that act as zinc ligands in β-lactamase II by differential tritium exchange

1979 ◽  
Vol 179 (3) ◽  
pp. 459-463 ◽  
Author(s):  
G S Baldwin ◽  
S G Waley ◽  
E P Abraham
Keyword(s):  
Bacillus Cereus ◽  
Binding Site ◽  
Beta Lactamase ◽  
Histidine Residues ◽  
Tryptic Digest ◽  
Beta Lactamase Ii

1. Four histidine-containing peptides have been isolated from a tryptic digest of the Zn2+-requiring beta-lactamase II from Bacillus cereus. One of these peptides probably contains two histidine residues. 2. The presence of one equivalent of Zn2+ substantially decreases the rate of exchange of the C-2 proton in at least two and probably three of the histidine residues of these peptides for solvent 3H. 3. It is concluded that peptides containing at least two of the three histidine residues acting as Zn2+ ligands at the tighter Zn2+-binding site of beta-lactamase II have been identified.

scholarly journals Identification of an essential glutamic acid residue in β-lactamase II from Bacillus cereus

1986 ◽  
Vol 233 (2) ◽  
pp. 465-469 ◽  
Author(s):  
C Little ◽  
E L Emanuel ◽  
J Gagnon ◽  
S G Waley
Keyword(s):  
Glutamic Acid ◽  
Bacillus Cereus ◽  
Gel Filtration ◽  
Thiol Group ◽  
Water Soluble ◽  
Beta Lactamase ◽  
Pepsin Digestion ◽  
Early Stages ◽  
Glutamic Acid Residue ◽  
Beta Lactamase Ii

Beta-Lactamase II from Bacillus cereus was readily inactivated by incubation at pH 4.75 with a water-soluble carbodiimide plus a suitable nucleophile. In the early stages of the reaction, 1 equivalent of nucleophile was incorporated/equivalent of enzyme, whereas during the later stages a second equivalent of nucleophile was also incorporated. This latter process correlated with the blocking of the enzyme's single thiol group. Enzyme inactivated in the presence of the coloured nucleophile N-(2,4-dinitrophenyl)ethylenediamine was fragmented by pepsin digestion, and coloured peptides were isolated by gel filtration and h.p.l.c. Two major peptides, representing 52% of the incorporated label, were isolated and sequenced. Both peptides contained the incorporated label on glutamic acid-37, and it is concluded that this latter residue represents a catalytically essential carboxylic residue in beta-lactamase II.

Interaction of β-lactamases I and II from Bacillus cereus with semisynthetic cephamycins. Kinetic studies

1991 ◽  
Vol 279 (1) ◽  
pp. 111-114 ◽  
Author(s):  
J Martin Villacorta ◽  
P Arriaga ◽  
J Laynez ◽  
M Menendez
Keyword(s):  
Bacillus Cereus ◽  
Kinetic Studies ◽  
Beta Lactamase ◽  
Beta Lactam ◽  
Beta Lactamases ◽  
Class A ◽  
Rate Determining Step ◽  
Class B ◽  
Lactam Ring ◽  
Beta Lactamase Ii

The influence of C-6 alpha- or C-7 alpha-methoxylation of the beta-lactam ring in the catalytic action of class A and B beta-lactamases has been investigated. For this purpose the kinetic behaviour of beta-lactamases I (class A) and II (class B) from Bacillus cereus was analysed by using several cephamycins, moxalactam, temocillin and related antibiotics. These compounds behaved as poor substrates for beta-lactamase II, with high Km values and very low catalytic efficiencies. In the case of beta-lactamase I, the substitution of a methoxy group for a H atom at C-7 alpha or C-6 alpha decreased the affinity of the substrates for the enzyme. Furthermore, the acylation of cephamycins was completely blocked, whereas that of penicillins was slowed down by a factor of 10(4)-10(5), acylation being the rate-determining step of the process.

scholarly journals Kinetics of inactivation of β-lactamase I by 6 β-bromopenicillanic acid

1980 ◽  
Vol 187 (3) ◽  
pp. 797-802 ◽  
Author(s):  
V Knott-Hunziker ◽  
B S Orlek ◽  
P G Sammes ◽  
S G Waley
Keyword(s):  
Bacillus Cereus ◽  
Rate Constant ◽  
Active Site ◽  
Beta Lactamase ◽  
Beta Lactam ◽  
Rate Determining Step ◽  
Competitive Model ◽  
Lactam Ring ◽  
Acid Inactivation ◽  
Kinetics Of

The kinetics of the inactivation of beta-lactamase I from Bacillus cereus 569 by preparations of 6 alpha-bromopenicillanic acid showed unexpected features. These can be quantitatively accounted for on the basis of the inactivator being the epimer, 6 beta-bromopenicillanic acid. At pH 9.2, the rate-determining step in the inactivation is the formation of the inactivator. When pure 6 beta-bromopenicillanic acid is used to inactivate beta-lactamase I, simple second-order kinetics are observed. The inactivated enzyme has a new absorption peak at 326 nm. The rate constant for inactivation has the same value as the rate constant for appearance of absorption at 326 nm; the rate-determining step may thus be fission of the beta-lactam ring of 6 beta-bromopenicillanic acid. Inactivation is slower in the presence of substrate, and the observed kinetics can be quantitatively accounted for on a simple competitive model. The results strongly suggest that inactivation is a consequence of reaction at the active site.

Cryoenzymology of Bacillus cereus .beta.-lactamase II

Biochemistry ◽  
1985 ◽  
Vol 24 (24) ◽  
pp. 6876-6887 ◽  
Author(s):  
Roy Bicknell ◽  
Stephen G. Waley
Keyword(s):  
Bacillus Cereus ◽  
Beta Lactamase ◽  
Beta Lactamase Ii
Sign in / Sign up

Export Citation Format

Share Document