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Biomedical subjects

J R Knowles

Publications and source records attributed to J R Knowles.

At least 109 records · Page 6Linked to original sources
PubMed1982

Targeted point mutation that creates a unique Eco RI site within the signal codons of the beta-lactamase gene without altering enzyme secretion or processing.

A method has been developed for constructing site-specific mutations by using a strongly selectable marker on which to "piggy-back" a desired mutation that may be phenotypically silent. Using this approach, a new unique Eco RI restriction site has been generated at the beginning of the signal codons of the beta-lactamase gene of the plasmid pBR322. The consequential alteration of the second amino acid of the signal from Ser to Arg has no effect on either the transport or the processing of the beta-lactamase.

Base Sequence↗
PubMed1982

Inhibition of the RTEM beta-lactamase from Escherichia coli. Interaction of the enzyme with derivatives of olivanic acid.

From chemical and kinetic studies of the interaction of the RTEM beta-lactamase from Escherichia coli with three derivatives of olivanic acid, MM22382 (1), MM13902 (2), and MM4550 (3), a mechanism for the inhibition of the enzyme by these compounds is proposed: the interaction proceeds by formation of an acyl-enzyme, the delta 2-pyrroline, which may either deacylate or undergo tautomerization to the more tightly bound delta 1-pyrroline. The ability of olivanic acids to inhibit the enzyme thus depends on the partitioning of the acyl-enzyme to the delta 1-pyrroline ( a process that competes with the normal hydrolytic pathway) and on the rate of regeneration of free enzyme from this complex.

Anti-Bacterial Agents↗
PubMed1982

The fate of the hydrogens of phosphoenolpyruvate in the reaction catalyzed by 5-enolpyruvylshikimate-3-phosphate synthase. Isotope effects and isotope exchange.

The condensation reaction of phosphoenolpyruvate and shikimate 3-phosphate catalyzed by 5-enolpyruvylshikimate-3-phosphate synthase is thought to proceed by an addition-elimination mechanism in which C-3 of phosphoenolpyruvate transiently becomes a methyl group in the enzyme-bound intermediate. Results obtained from reactions conducted in H2O, 2H2O, and 3H2O, using unlabeled, [3-2H2]-, or [3-3H,2H]phosphoenolpyruvate, are consistent with the addition-elimination pathway and show that the transient methyl group rotates rapidly. There is substantial discrimination against heavy hydrogen isotopes in both the protonation and deprotonation steps. These results demonstrate the feasibility of determining the stereochemical course of the synthase reaction.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMed1981

Stereochemistry of phospho transfer catalyzed by bovine liver acid phosphatase.

The hydrolysis of phosphoric monoesters by acid phosphatases is thought to proceed via the formation of a phosphoenzyme intermediate, but no stereochemical evidence exists on this point. We have carried out the transphosphorylation from phenyl (R)-[16O, 17O, 18O] phosphate to (S)-propane-1,2-diol in the presence of homogeneous bovine liver acid phosphatase, and have found that the reaction proceeds with greater 90% overall retention of configuration at phosphorus. This stereochemical course of phospho transfer during the enzyme-catalyzed reaction of the phosphoric monoester is consistent with a double displacement mechanism in which each step proceeds with inversion of the stereochemistry at phosphorus, and is similar to the behavior of the bacterial alkaline phosphatase.

Acid Phosphatase↗
PubMed1981

The stereochemical course of the reaction catalyzed by creatine kinase.

Adenosine [gamma-(S)-16O, 17O, 18O]triphosphate has been used as a substrate in the reaction catalyzed by creatine kinase, and the configuration at phosphorus in the product [16O, 17O, 18O]phosphocreatine has been determined. The reaction proceeds with inversion of the configuration, consistent with the emerging pattern of behavior among the phosphokinases. This result, coupled with recently published data from NMR, ESR, and infrared studies of ternary and quaternary complexes od creatine kinase with its substrates and inhibitors, defines the enzyme-catalyzed transphosphorylation as an associative in-line transfer of the phosphoryl group between the bound substrates.

Adenosine Triphosphate↗
PubMed1981

Penicillanic acid sulfone: an unexpected isotope effect in the interaction of 6 alpha- and 6 beta-monodeuterio and of 6,6-dideuterio derivatives with RTEM beta-lactamase from Escherichia coli.

Penicillanic acid sulfone (1) is both a substrate and an inactivator of the RTEM beta-lactamase. About 7000 hydrolytic events occur before enzyme inactivation. The 6,6-dideuterio sulfone shows a 3-fold acceleration of both the hydrolysis reaction and the enzyme inactivation. The kinetic and spectroscopic results are nicely accommodated by a scheme in which a transiently stable intermediate is formed in an isotopically sensitive step. The deuterated material partitions less readily toward this transiently stable intermediate by virtue of a primary isotope effect, and more enzyme is then available for the hydrolysis and inactivation pathways. Use of the stereospecifically monodeuterated sulfones shows that the 6 beta hydrogen is preferentially abstracted in the formation of the transiently stable intermediate and allows a detailed picture of the interaction of the sulfone and the beta-lactamase to be drawn. The crystal structures of both the labeled and unlabeled compounds are reported.

Deuterium↗
PubMed1981

Penicillanic acid sulfone: interaction with RTEM beta-lactamase from Escherichia coli at different pH values.

The interaction of the sulfone of penicillanic acid with the TEM-2 beta-lactamase from Escherichia coli has been investigated as a function of pH between pH 7.0 and 9.6. The first-formed acyl-enzyme suffers one of three fates: deacylation, tautomerization to a bound enamine that transiently inhibited the enzyme, and a process (possibly transimination) that leads to enzyme inactivation. The observed changes in ultraviolet absorbance are consistent with the initially observed product of deacylation being the enamine tautomer (4) of the imine from malonsemialdehyde and penicillamine sulfinate. The same enamine can be generated nonenzymically from the sulfone at high pH. The transiently inhibited enzyme appears to be the same enamine attached to the enzyme by an ester linkage. The rather complex kinetic behavior can be deconvuluted by exploiting the effect of pH on the partitioning of the acyl-enzyme between deacylation and the transiently inhibited form of the enzyme. The pathways followed by penicillanic acid sulfone provide a model for the behavior of a number of other reagents that inactivate the beta-lactamase.

Escherichia coli↗
PubMed1981

Inactivation of RTEM beta-lactamase from Escherichia coli by clavulanic acid and 9-deoxyclavulanic acid.

The interaction of the TEM-2 beta-lactamase with 9-deoxyclavulanic acid (3) and with both extensively labeled (2) and specifically labeled (1) clavulanic acid has been studied. The close similarity between 9-doexyclavulanate and clavulanate in kinetics, spectroscopic, and protein chemical terms show that the allyl alcohol group of clavulanate is irrelevant to its action as a beta-lactamase inactivator. Use of the radiolabeled samples of clavulanate shows that, of three irreversibly inactivated forms of the enzymes, two contain the whole clavulanate skeleton and the third only retains the carbon atoms of the original beta-lactam ring. These findings allow the complex interaction between clavulanic acid and the beta-lactamase to be defined more narrowly in chemical terms.

Clavulanic Acid↗
PubMed1981

Inactivation of the RTEM beta-lactamase from Escherichia coli. Interaction of penam sulfones with enzyme.

The characteristics of the reaction of a number of mechanism-based inactivators of the RTEM beta-lactamase have suggested that a common mechanistic pathway may be followed by many of these compounds. These ideas have been tested by the synthesis and evaluation of some penam sulfones as beta-lactamase inactivators. The sulfones of poor beta-lactamase substrates are, as predicted, potent inactivators of the enzyme. A unique serin residue (Ser-70) is labeled by quinacillin sulfone, and it is likely that this serine acts nucleophilically in the normal hydrolytic reaction of the beta-lactamase to form an acyl-enzyme intermediate.

Amino Acids↗
PubMed1981

Inhibition of the RTEM beta-lactamase from Escherichia coli. Interaction of enzyme with derivatives of olivanic acid.

The interaction of the RTEM beta-lactamase with two derivatives of olivanic acid has been studied. The compound MM22382 (1) behaves simply as a good substrate for the enzyme and is a relatively ineffective inhibitor. In contrast, the sulfate ester MM13902 (2) is a poor substrate and an excellent inhibitor of the enzyme. The inhibition derives from a branching of the normal hydrolytic pathway of the enzyme. At long times, all the catalytic activity of the enzyme returns. Free sulfate ion is not produced during the interaction with the enzyme, which rules out a mechanistic pathway involving beta elimination between C-6 and C-8. The validity of a number of alternative schemes is assessed.

Anti-Bacterial Agents↗
PubMed1980

Photogenerated reagents for membranes: selective labeling of intrinsic membrane proteins in the human erythrocyte membrane.

1-[3H]Spiro[adamantane-4,4'-diazirine], a lipophilic, photoactivatable reagent designed to label those segments of intrinsic proteins that lie within the lipid bilayer of biological membranes, has been evaluated. The reagent labels the intrinsic proteins of human erythrocyte membranes far more heavily than it labels the extrinsic proteins. This result, together with a detailed analysis of the label distribution in several well-characterized membrane proteins [Goldman, D.W., Pober, J.S., White, J., & Bayley, H. (1979) Nature (London) 280, 841], demonstrates that labeling with adamantanediazirine is a convenient and rapid method both for distinguishing intrinsic from extrinsic membrane proteins and for locating within intrinsic proteins those amino acid residues that are in contact with the hydrocarbon core of the lipid bilayer.

Adamantane↗
PubMed1980

beta-Lactamase proceeds via an acyl-enzyme intermediate. Interaction of the Escherichia coli RTEM enzyme with cefoxitin.

The use of cefoxitin, a poor substrate of the RTEM beta-lactamase, has allowed the kinetic and spectroscopic characterization of a covalent acyl-enzyme intermediate in the enzyme-catalyzed reaction. The rate of reappearance of catalytic activity in an enzyme sample diluted from an incubation with cefoxitin is nearly identical with the observed Kcat. Burst kinetics are observed with this substrate, consistent with the rate-limiting deacylation of the cefoxitinoyl-enzyme. That the reaction intermediate involves a covalent link between enzyme and substrate was shown by gel filtration after rapid denaturation of an enzyme-[14C]cefoxitin reaction at the steady state. Fourier transform infrared measurements indicate that the intermediate is an acyl-enzyme involving a hydroxyl group of the beta-lactamase. The evident relationship between the acylation-deacylation sequence of the beta-lactamases and the acylation reaction suffered by the D-Ala-D-Ala-carboxypeptidases is discussed.

Binding Sites↗
PubMed1980

Photoaffinity labeling of lactate dehydrogenase by the carbene derived from the 3-diazirino analogue of nicotinamide adenine dinucleotide.

The 3-diazirino analogue of NAD+, DAD+, in which the diazirine group replaces the carboxamide in an almost isosteric substitution, has been synthesized as a photoaffinity reagent for dehydrogenases. With lactate dehydrogenase, the Kdiss is 4 mM (compare the NAD+ Kdiss of 0.6 mM). On photolysis, three types of interaction can be discerned and separately quantitiated by using [3H]DAD+: noncovalent binding of the photoproducts of DAD+, which is removed by protein denaturation; nonspecific covalent labeling, which is eliminated by the presence of a scavenger such as glutathione; specific covalent labeling at the active site, which is prevented by competition from the natural ligands. The photolabeling efficiency (sites covalently labeled/sites initially occupied) is approximately 0.3. It is evident that the carbene generated from DAD+ is more effective than that from the more bulky 3-diazoacetate or than the less reactive nitrene derived from the 3-azido-NAD+ analogue.

Affinity Labels↗
PubMed1980

Beta-lactamase inactivation by mechanism-based reagents.

The mechanistic pathway followed by the E. coli RTEM beta-lactamase has been studied with a view to clarifying the mode of action of a number of recently discovered inactivators of the enzyme. There is clear evidence that the beta-lactamase-catalysed hydrolysis of the 7-alpha-methoxycephem, cefoxitin, proceeds via an acyl-enzyme intermediate. An analysis of the inactivation reactions of all the known beta-lactam derivatives that result in irreversible loss of enzyme activity permits the identification of three structural features required for a beta-lactamase inactivator. The application of these principles suggests a new group of mechanism-based inactivators of the enzyme: the sulphones of N-acyl derivatives of 6-beta-aminopenicillanic acid that are themselves poor substrates for the enzyme. These sulphones are powerful inactivators of the beta-lactamase.

Anti-Bacterial Agents↗
PubMed1980

Phosphoglycerate mutases: stereochemical course of the phosphoryl group transfers catalyzed by the cofactor-dependent enzyme from rabbit muscle and the cofactor-independent enzyme from wheat germ.

2-[(R)-16O,17O,18O]Phospho-D-glycerate has been synthesized and used to determine the stereochemical course of each of the two mechanistic classes of phosphoglycerate mutases. The enzyme from rabbit muscle requires 2,3-bis-phospho-D-glycerate as a cofactor and catalyzes an intermolecular phosphoryl group transfer reaction. The enzyme from wheat germ requires no cofactor and catalyzes an intramolecular transfer of the phosphoryl group. We have shown that the reaction catalyzed by each of these enzymes proceeds with overall retention of the configuration at phosphorus. This stereochemical result is consistent with a double-displacement pathway involving a single phosphorylenzyme, for each of the catalyzed reactions.

Animals↗