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G Lowe

Publications and source records attributed to G Lowe.

At least 127 records · Page 7Linked to original sources
PubMed1982

The stereochemical course of phosphoryl transfer catalysed by glucose 6-phosphatase.

Rat liver microsomal glucose 6-phosphatase catalyses phosphoryl transfer between D-glucose 6-[(R)-16O,17O,18O]phosphate and D-glucose with retention of configuration at the phosphorus atom. Since individual phosphoryl-transfer steps appear in general to occur with inversion of configuration, this observation is most simply interpreted in terms of a double-displacement mechanism with a phosphoryl-enzyme intermediate. Such an intermediate has been proposed previously from kinetic and 32P-labelling experiments.

Animals↗
PubMed1982

The stereochemical course of phosphoryl transfer catalysed by glucokinase.

Adenosine 5'-[gamma(S)-16O,17O,18O]triphosphate has been used to determine the stereo-chemical course of phosphoryl transfer catalysed by rat liver glucokinase. The chirality of the product, D-glucose 6-[16O,17O,18O]phosphate was analysed by 31P n.m.r. spectroscopy. The reaction proceeds with inversion of configuration at phosphorus. The simplest interpretation of this result, which is the same as that observed with yeast hexokinase [Lowe & Potter (1981) Biochem. J. 199, 277-233], is that the phosphoryl group is transferred between MgATP2- and glucose in the ternary complex by an 'in-line' mechanism. It accords with the veiw that the kinetic differences between glucokinase and the other hexokinases arise from differences in rate constants and not from any fundamental differences in chemical mechanism.

Adenosine Triphosphate↗
PubMed1981

A stereochemical investigation of phosphoryl transfer catalysed by phosphoglucomutase by the use of alpha-D-glucose 1-[(S)-16O,17O,18O]phosphate.

Rabbit muscle phosphoglucomutase converts alpha-D-glucose 1-[(S)-16O,17O,18O]phosphate into D-glucose 6-[16O,17O,18O]phosphate, which is shown by 31P n.m.r. spectroscopy, after cyclization and methylation, to have the (S)-configuration at the phosphorus atom. Since phosphoglucomutase is known to catalyse phosphoryl transfer by way of a phospho-enzyme intermediate, and since individual phosphoryl-transfer steps appear in general to occur with inversion of configuration, this observation is most simply interpreted in terms of a double-displacement mechanism with two phosphoryl-transfer steps.

Animals↗
PubMed1981

The stereochemical course of phosphoryl transfer catalysed by Bacillus stearothermophilus and rabbit skeletal-muscle phosphofructokinase with a chiral [16O,17O,18O]phosphate ester.

Bacillus stearothermophilus and rabbit skeletal-muscle phosphofructokinases catalyse the transfer of the chiral [16O,17O,18O]phosphoryl group from D-fructose 1[(S)-16O,17O,18O],6-bisphosphate to ADP with inversion of configuration at the phosphorus atom. D-Fructose 1[(S)-16O,17O,18O],-bisphosphate was synthesized in situ from sn-glycerol 3[(S)-16O,17O,18O]phosphate. The simplest interpretation of these results is that the phosphoryl group is transferred between substrates in the enzyme substrate ternary complexes by an 'in-line' mechanism.

Adenosine Triphosphate↗
PubMed1981

The stereochemical course of hydrolysis catalysed by snake venom 5'-nucleotide phosphodiesterase.

Adenosine 5'-(S)-[16O,17O,18O]phosphate was pyrophosphorylated by the combined action of adenylate kinase and pyruvate kinase. The isotopomers of adenosine 5'-[alpha-16O,17O,18O]triphosphate were hydrolysed by venom 5'-nucleotide phosphodiesterase (Crotalus adamanteus) in H2(17)O. Analysis by 31P nuclear magnetic resonance spectroscopy of the resulting adenosine 5'-[16O,17O,18O]phosphate, after cyclization and esterification, showed that the hydrolysis occurs with retention of configuration at phosphorus. The most likely explanation of this observation is that the enzymic hydrolysis involves a double displacement at phosphorus with a covalent nucleotidyl--enzyme intermediate on the reaction pathway.

Adenosine Monophosphate↗
PubMed1981

The stereochemical course of yeast hexokinase-catalysed phosphoryl transfer by using adenosine 5'[gamma(S)-16O,17O,18O]triphosphate as substrate.

Adenosine 5'[gamma(S)-16O, 17O, 18O]triphosphate has been synthesized and used to determine the stereochemical course of phosphoryl transfer catalysed by yeast hexokinase. The chirality at phosphorus of the D-glucose 6-[16O,17O,18O]phosphate formed was analysed, after cyclization and methylation, by 31P n.m.r. spectroscopy. The phosphoryl transfer was found to occur with inversion of configuration, with a stereoselectivity in excess of 94%. The simplest interpretation of this result is that the phosphoryl group is transferred between substrates in the enzyme-substrate ternary complex by an 'in line' mechanism.

Adenosine Triphosphate↗
PubMed1981

The stereochemical course of phosphoryl transfer catalysed by polynucleotide kinase (bacteriophage-T4-infected Escherichia coli B).

Polynucleotide kinase (bacteriophage-T4-infected Escherichia coli B) catalyses the transfer of the [gamma-16O,17O,18O]phosphoryl group from 5'[gamma(S)-16O,17O,18O]ATP to 3'-AMP with inversion of configuration at the phosphorus atom. The simplest interpretation of this observation is that the [gamma-16O,17O,18O]phosphoryl group is transferred directly from ATP to the co-substrate by an 'in-line' mechanism.

Adenosine Triphosphate↗
PubMed1981

Stereochemistry of phosphoryl transfer.

A general method has been developed for the synthesis of chiral [16O,17O,18O]phosphate monoesters of known absolute configuration. An analytic method for determining the absolute configuration of chiral phosphate esters has also been developed, which is based on the isotope effects of 17O and 18O at phosphorus in the 31P nuclear magnetic resonance spectrum. These methods have shown that phosphoryl transfer catalysed by hexokinase, phosphofructokinase and pyruvate kinase occurs with inversion of configuration. This is most simply interpreted as an "in-line' transfer of the phosphoryl group between substrates in the enzyme-substrate ternary complex.

Animals↗
PubMed1981

Mechanism of activation of bicarbonate ion by mitochondrial carbamoyl-phosphate synthetase: formation of enzyme-bound adenosine diphosphate from the adenosine triphosphate that yields inorganic phosphate.

The mechanism of the reaction catalyzed by rat liver mitochondrial carbamoyl-phosphate synthetase has been studied by using [beta-18O2]ATP and HC18O-3, monitoring the isotopic composition of adenosine triphosphate (ATP) and inorganic phosphate (Pi) by high-resolution 31P NMR spectroscopy. In the presence of both HCO3- and acetylglutamate, the enzyme catalyzes the exchange of oxygen atoms between the beta, gamma bridging and the beta nonbridging positions of ATP. Addition of NH3 stops the exchange, Pi released by the ATPase activity of the enzyme in the absence of NH3 contains one oxygen atom from HC18O3- but there is no incorporation of 18O into ATP. There is no significant incorporation of [14C]ADP or 32Pi into ATP. It is concluded that in the enzyme-ATPA.HCO30.ATPB complex formed in the presence of ATP and HCO3- there is reversible transfer of the gamma-PO3 group of ATPA (the molecule that yields Pi) to HCO3- without dissociation of products. The beta-PO3 of the enzyme-bound ADP that is formed can rotate. Virtually all of the complex appears to be in the form in which ATPA is cleaved, but in the absence of NH3, ATP is reconstituted and dissociates from the complex on at least 75% of the occasions. On the remainder, the carbonyl phosphate is cleaved in an irreversible process that yields Pi and a low-energy form of carbonic acid (probably HCO3-). NH3 reacts rapidly and irreversibly with the complex, and at saturation the rate (greater than 10 times the rate of Pi release in the absence of NH3) is sufficient to prevent dissociation of ATPA. In the absence of HCO3- an enzyme-ATPA.ATPB complex is formed, but cleavage of the bond between beta, gamma bridging oxygen and P gamma of ATPA does not occur.

Adenosine Diphosphate↗
PubMed1980

Evidence for an associative mechanism in the phosphoryl transfer step catalyzed by rabbit muscle creatine kinase.

Creatine kinase does not catalyze the scrambling of 18O in adenosine 5'-[alpha beta-18O, beta-18O2]triphosphate in the absence of creatine, in the presence of L-arginine or taurocyamine (competitive inhibitors of creatine), or in the presence of poor substrates where single turnover experiments were performed. In order to support this prima facie evidence for an associative mechanism of phosphoryl transfer, an investigation was undertaken of 1-carboxymethyl-2-aminoimidazole, a new substrate analogue of creatine. This analogue has a binding constant for rabbit muscle creatine kinase similar to creatine and 1-carboxymethyl-2-iminoimidazolidine, but the initial rate of phoshorylation by MgATP in the presence of creatine kinase is almost 5 orders of magnitude slower. The phosphorylation product, assigned the structure 1-carboxymethyl-2-imino-3-phospho-4-imidazoline is also a poor substrate for the phosphorylation of MgADP by creatine kinase. These observations can be accounted for by an associative SN2(P) mechanism of phosphoryl transfer and by a microenvironment of the enzyme-bound creatine (or creatine analogue) which lowers the pKa of the guanidino group by several pH units compared with that in aqueous solution.

Animals↗
PubMed1980

Evidence against a step-wise mechanism for the fumarase-catalysed dehydration of (2S)-malate.

Fumarase does not catalyse 18O exchange between (2S,3R)-tartrate and solvent water, nor does it catalyse 2H or 3H exchange into (2S)-fluorosuccinate from isotopically labelled water. Both of these substrate analogues are good competitive inhibitors of fumarase. This lack of isotopic exchange provides prima facie evidence against the stepwise carbenium ion and carbanion mechanisms. This, together with evidence from the literature, suggests that the fumarase-catalysed dehydration of (2S)-malate occurs by a concerted mechanism in which breaking of the C--OH bond is much further advanced than that of the C--H bond in the transition state.

Animals↗
PubMed1979

The stereochemical course of amino acid activation by methionyl- and tyrosyl-tRNA synthetases.

Stereochemical analysis has long been recognised as a powerful tool for elucidating the mechanisms of chemical and enzyme-catalysed reactions. Although much is known about the stereochemical course of reactions at saturated carbon, phosphate and thiophosphate esters whose ligands to phosphorus are also tetrahedrally disposed, are capable in principle of revealing sterochemical information about events at the active site of enzymes that transform such substrates. Nucleotidyl transferases are a group of enzymes which in general selectively use one of the diastereoisomers of a nucleoside 5'(1-thiotriphosphate), such as isomers A and B of adenosine 5'(1-thiotriphosphate), designated ATP alpha S-A and ATP alpha S-B, and allow investigation of the stereochemical course of nucleotidyl transfer. We have developed a simple method based on 31P nuclear magnetic resonance spectroscopy for determining the stereochemical course of these reactions, and using this method show here that the nucleotidyl transfer step in two aminoacyl-tRNA synthetases from Escherichia coli occurs with inversion of configuration at phosphorus. These observations greatly constrain the mechanistic possibilities for these enzymes, and are interpreted most simply as a direct 'in line' transfer from ATP to the amino acid.

Amino Acyl-tRNA Synthetases↗
PubMed1979

Evidence for hemiacetal formation between N-acyl-L-phenylalaninals and alpha-chymotrypsin by cross-saturation nuclear magnetic resonance spectroscopy.

N-Acetyl-L-phenylalaninal exists predominantly in its hydrated form in aqueous solution, but the aldehyde and not the hydrate is shown by nuclear magnetic resonance (NMR) spectroscopy to be the effective inhibitor of alpha-chymotrypsin. NMR spectroscopy also indicates that the initial alpha-chymotrypsin-N-acetyl-L-phenylalaninal complex is in equilibrium with a hemiacetal formed between the aldehyde and the active site serine residue. The rate of the latter equilibration is slow on the NMR time scale but the hemiacetal can be detected by cross-saturation NMR spectroscopy. N-Benzoyl-L-phenylalaninal is a more potent inhibitor of alpha-chymotrypsin than the N-acetyl derivative and both the formation of the enzyme-inhibitor complex and the hemiacetal are slow on the NMR time scale, but the hemiacetal in the enzyme can be detected by cross-saturation NMR spectroscopy. The N-acyl-L-phenylalaninals also bind to N-methylhistidinyl-57-alpha-chymotrypsin, but clear evidence for hemiacetal formation was not obtained by cross-saturation NMR spectroscopy either because the hemiacetal was not formed or more probably because the rate of dissociation was slow compared with the rate of relaxation of the hemiacetal proton. The dissociation constant of N-benzoyl-L-phenylalaninal to dehydroalaninyl-195-alpha-chymotrypsin was found to be high relative to the dissociation constant to native alpha-chymotrypsin, supporting the NMR evidence that a hemiacetal with the Ser-195 is formed on association of N-benzoyl-L-phenylalaninal with alpha-chymotrypsin.

Acetals↗
PubMed1978

Conversion of the active-site cysteine residue of papain into a dehydro-serine, a serine and a glycine residue.

Photolysis of papain which had been inhibited with 2-bromo-2',4'-dimethoxyacetophenone regenerated papain, but also formed [deltaSer25]-papain (i.e. papain in which the active-site cysteine residue 25 was replaced by dehydroserine) via the intermediate dehydrocysteine analogue, [deltaCys25]-papain. Reduction with sodium borohydride gave [Ser25]papain. Both [Ser25]papain and [deltaSer25]-papain had binding properties similar to those of papain, but were devoid of enzymic activity. Their fluorescence properties were also investigated. Incubation of [deltaSer25]papain at pH 9.0 gave [Gly25]papain.

Acetophenones↗
PubMed1977

Inhibition of fructose-1,6-bisphosphate aldolase from rabbit muscle and Bacillus stearothermophilus.

Phosphoglycollohydroxamic acid and phosphoglycollamide are inhibitors of rabbit muscle fructose-1,6-bisphosphate aldolase. The binding dissociation constants determined by enzyme inhibition and protein fluorescence quenching suggest that two distinct enzyme inhibitor complexes may be formed. The binding dissociation constants of the two inhibitors to Bacillus stearothermophilus cobalt (II) fructose-1,6-bisphosphate aldolase have also been determined. The hydroxamic acid is an exceptionally potent inhibitor (Ki = 1.2 nM) probably due to direct chelation with Co(II) at the active site. The inhibition, however, is time-dependant and the association and dissociation constants have been estimated. Ethyl phosphoglycollate irreversibly inhibits rabbit muscle fructose-1,6-bisphosphate aldolase in the presence of sodium borohydride, presumably by forming a stable secondary amine through the active-site lysine reside. A new condensation assay for fructose-1,6-bisphosphate aldolases has been developed which is more sensitive than currently used assay procedures.

Animals↗