Improved conditions for the removal of 2-oxoacyl groups from the N-terminus of proteins.

Proteins with R-CO-CO-NH- at the N-terminus, rather than the usual R-CH(-NH3+)-CO-NH-, are produced by non-enzymic transamination and also occur in the pyruvoyl enzymes. The oxoacyl group may be specifically removed from a model peptide, in yields of 70-80%, by treating them in 0.1 M phosphate buffer at 37 degreesC for 24 h with 25 mM of the N-phosphonomethyl derivative of phenylene-1,2-diamine. This provides mild conditions for the stepwise removal of N-terminal residues without denaturation.

[Reactions of decarboxylated and side transamination during interaction of glutamate decarboxylase from Escherichia coli with substrate analogs, modified through C3 and C4 atoms]. / Reaktsii dekarboksilirovaniia i pobochnogo transaminirovaniia pri vzaimodeistvii glutamatdekarboksilazy iz Escherichia coli s analogami substrata, modifitsirovannymi po atomam C3 i C4.

The interaction of glutamate decarboxylase with the aspartate and glutamate analogues modified at C3 and C4 was studied. 3-Arsonoalanine, 3-phosphonoalanine, 2-amino-4-arsonobutyric acid, 2-amino-4-phosphonobutyric acid, a mixture of diastereoisomers of 4-(methylthio) glutamic acid and erythro-4-(methylthio) glutamic acid were shown to be poor substrates for the enzyme. Their decarboxylation was accompanied by transamination of the coenzyme (PLP) to pyridoxamine phosphate (PMP) which reversibly inactivated the enzyme. With arsonoalanine only part of PLP was converted into PMP and another part irreversibly formed a complex. 4-(Methylsulfonyl)-L-glutamic and 4-[(phenyl)(hydroxy)phosphoryl]-L-glutamic acids did not react with the glutamate decarboxylase.

Arsenite release on enzymic transformation of arsonomethyl substrate analogues: a potentially lethal synthesis by glycerol-3-phosphate dehydrogenase.

The isosteric arsenical analogue of glycerol 3-phosphate, 3,4-dihydroxybutylarsonic acid, is a good substrate for rabbit muscle glycerol-3-phosphate dehydrogenase. Its oxidation is accompanied by release of arsenite. This release seems to be due to a spontaneous elimination of arsenite by 3-oxoalkylarsonic acids, as it is also observed in (1) the oxidation of 3-hydroxypropylarsonic acid by yeast alcohol dehydrogenase, (2) treatment of 3,4-dihydroxybutylarsonic acid with periodate and (3) nonenzymic transamination of the glutamate analogue 2-amino-4-arsonobutyric acid. Enzymic formation of 3-oxoalkylarsonic acids in cells can therefore be lethal, as arsenite is poisonous to most organisms because of its high affinity for dithiols such as dihydrolipoyl groups.

The interaction of glutamate decarboxylase from Escherichia coli with substrate analogues modified at C-3 and C-4.

The interaction of glutamate decarboxylase with the aspartate analogues 3-arsonoalanine and 3-phosphonoalanine, with the glutamate analogues 2-amino-4-arsonobutyric acid and 2-amino-4-phosphonobutyric acid, and with 4-(methylthio)-L-glutamic acid, both as a mixture of diastereoisomers and as the (2S,4R)-form, was studied. All these analogues were poor substrates for the enzyme and only weak inhibitors. Their decarboxylation was accompanied by transamination of the enzyme-bound pyridoxal phosphate (PLP) to pyridoxamine phosphate (PMP), thus inactivating the decarboxylase. With arsonoalanine only part of the PLP was converted into PMP.

A rapid spectrophotometric estimation of individual phosphates and phosphonates: its application to chromatography of sugar phosphates and their phosphonate analogues.

A compound containing the -PO3H2 group (phosphoric acid, one of its monoesters, or an alkylphosphonic acid) may be rapidly assayed by the decrease it produces in the absorbance at 450 nm of a buffered acidic solution of Fe3+ and N(-3) [corrected]. The method has been used to follow chromatograms of sugar phosphates and their phosphonomethyl analogues.

The preparation and properties of bromoacetylphosphonic acid.

Bromoacetylphosphonic acid, Br-CH2-CO-PO3H2, was made by brominating dimethyl acetylphosphonate and de-esterifying with HBr. It proves to be a powerful alkylating agent, reacting rapidly with GSH, with a rate constant of about 6M(-1).s(-1) at pH6.

A synthesis of acylphosphonic acids and of 1-aminoalkylphosphonic acids: the action of pyruvate dehydrogenase and lactate dehydrogenase on acetylphosphonic acid.

Acylphosphonic acids, R-CO-PO(OH)2, have been synthesized by the steps [formula: see text] of which the last is new and provides a mild method for de-esterifying acylphosphonic acids. Their reductive amination gives a simple way of making 1-aminoalkylphosphonic acids. Acetylphosphonic acid inhibited NAD+ reduction by pyruvate with the pyruvate dehydrogenases from Escherichia coli and Bacillus stearothermophilus. The inhibition was competitive with pyruvate, with Ki of 6 microM for the E. coli enzyme (pyruvate Km 0.5 mM) and one of 0.4 mM of the B. stearothermophilus enzyme (pyruvate Km 0.1 mM). Acetylphosphonate and its monomethyl ester are substates for pig heart lactate dehydrogenase, with Km values of 15 mM and 10 mM respectively (pyruvate Km 0.05 mM) and specificity constants one thousandth that for pyruvate.

The synthesis of 3-phosphonoalanine, phosphonopyruvic acid and phosphonolactic acid. Scission of the C-P bond during diazotization of phosphonoalanine.

3-Phosphonoalanine has been made by the Strecker synthesis from phosphonoacetaldehyde, which is easily prepared from vinyl acetate. It gives phosphonopyruvate by transamination when treated with glyoxylate. Phosphonolactate, an analogue of phosphoglycerate, is prepared by reducing phosphonopyruvate. Diazotization of phosphonoalanine was investigated as a route for making phosphonolactate: addition of NaNO2 to the isoelectric form of phosphonoalanine gave much scission of the C-P bond with release of phosphate; addition of HBr prevented this release and gave largely the bromo acid. The supplement reports the synthesis of arsonolactate, a similar analogue, by treating chlorolactate with alkaline arsenite.

Application of the synthetic method to other amines.

Taurine and 2-aminoethylphosphonic acid were synthesized by the method of the main paper [Geoghegan & Dixon (1989) Biochem. J. 260, 295-296], i.e. by treating the corresponding halo compound with 2-aminoethanol and then with periodate.

The arsonomethyl analogue of adenosine 5'-phosphate. An uncoupler of adenylate kinase.

Adenosine was converted into the arsonomethyl analogue of AMP. The reactions used provide a general route for converting an alcohol, R-CH2-OH, into the arsonomethyl analogue, R-CH2-CH2-AsO3H2, of its phosphate, R-CH2-O-PO3H2. The analogue of AMP proves to be a substrate for rabbit adenylate kinase, which shows a limiting velocity with it of 1/17 that with AMP, a Michaelis constant raised 70-fold to about 10 mM, and hence a specificity constant lowered about 1200-fold. The product of transfer of a phospho group from ATP to the analogue is, like all anhydrides of arsonic acids, unstable to hydrolysis, and so breaks down to yield orthophosphate and regenerate the analogue. Hence adenylate kinase is converted into an ATPase by the presence of the analogue.

The arsonomethyl analogue of 3-phosphoglycerate.

4-Arsono-2-hydroxybutanoic acid, the analogue of 3-phosphoglycerate in which -CH2-AsO3H2 replaces -O-PO3H2, was synthesized. It proved to be a substrate for phosphoglycerate kinase. Its Michaelis constant was only slightly higher than that of the natural substrate, but its catalytic constant was about 1300 times smaller.

An arsenical analogue of adenosine diphosphate.

An analogue of ADP was made in which the terminal phosphono-oxy group, -O-PO(OH)2, has been replaced by the arsonomethyl group, -CH2-AsO(OH)2. This compound cannot form a stable analogue of ATP because anhydrides of arsonic acids are rapidly hydrolysed, so that any enzyme that phosphorylates ADP and accepts this analogue as a substrate should release orthophosphate in its presence. The analogue proves to be a poor substrate for 3-phosphoglycerate kinase (V/Km is diminished by a factor of 10(2)-10(3)) and a very poor substrate for pyruvate kinase (V/Km is diminished by a factor of 10(5)-10(6)). No substrate action was detected with adenyl kinase and creatine kinase.

Phosphonomethyl analogues of phosphate ester glycolytic intermediates.

Analogues of dihydroxyacetone phosphate and of 3-phosphoglycerate were made in which the phosphate group, -O-PO(3)H(2), is replaced by the phosphonomethyl group, -CH(2)-PO(3)H(2). The analogue of dihydroxyacetone phosphate is a substrate for aldolase and glycerol 1-phosphate dehydrogenase (Stribling, 1974), but not for triose phosphate isomerase. The analogue of 3-phosphoglycerate oxidizes NADH under the combined action of 3-phosphoglycerate kinase and glyceraldehyde 3-phosphate dehydrogenase if ATP is added. Thus four out of the five glycolytic enzymes tested handle the phosphonomethyl compounds like the natural phosphates.