Hydrolysis of Organophosphate Esters: Phosphotriesterase Activity of Metallo-beta-lactamase and Its Functional Mimics

The phosphotriesterase (PTE) activity of a series of binuclear and mononuclear zinc(II) complexes and metallo-beta-lactamase (m beta 1) from Bacillus cereus was studied. The binuclear complex 1, which exhibits good m beta 1 activity, shows poor PTE activity. In contrast,complex 2, a poor mimic of m beta 1, exhibits much higher activity than 1 The replacement of Cl- ligands by OH- is important for the high PTE activity of complex 2 because this complex does not show any catalytic activity in methanol. The natural enzyme m beta 1 from B. cereus is also found to be an inefficient catalyst in the hydrolysis of phosphotriesters. These observations indicate that the binding of beta-lactam substrates at the binuclear zinc(II) center is different from that of phosphotriesters. Furthermore, phosphodiesters, the products from the hydrolysis of triesters, significantly inhibit the PTE activity of m beta 1 and its functional mimics. Although the mononuclear complexes 3 and 4 exhibited significant m beta 1 activity, these complexes are found to be almost inactive in the hydrolysis of phosphotriesters. These observations indicate that the elimination of phosphodiesters from the reaction site is important for the PTE activity of zinc(II) complexes.

Beta-turns in nascent procollagen are sites of posttranslational enzymatic hydroxylation of proline

The selective hydroxylation of proline residues in nascent procollagen chains by prolyl hydroxylase (EC 1.14.11.2) can be understood in terms of the conformational feature of the -Pro-Gly-segments in linear peptides and globular proteins. The folded beta-turn conformation in such segments appears to be the conformational requirement for proline hydroxylation. The available data on the hydroxylation of native and synthetic substrates of prolyl hydroxylase are explained on the basis of the extent of beta-turn formation in them. Taken in conjunction with the conformational features of the hydroxyproline residue, our results bring out the conformational reason for the posttranslational proline hydroxylation which, it is proposed, leads to the "straightening" of the beta-turn segments into the linear triple-helical conformation.

Aspects of tautomerism. 7. Study of keto participation in alkaline hydrolysis of normal esters of .gamma.-keto acids

Study of the alkaline hydrolysis of a number of variously substituted normal o-benzoylbenzoic esters has been reported. Although carbonyl-assisted hydrolysis is the general rule, in compounds containing strongly electron-donating groups, the ester function is directly attacked. The cause of rate enhancement in carbonyl-assisted hydrolysis and in greater detail the case of 6-substituted derivatives are discussed. It is shown that the carbonyl-assisted hydrolyses are characterized by decreased sensitivity to leaving-group structure. The implications of this result are pointed out.

Do the Hydrolysis Products, Methylamine and N,N'-Dimethylurea, Play any Role in the Methyl Isocyanate-Induced Haematological and Biochemical Changes in Rabbits?

The subcutaneous administration of methyl isocyanate (MIC) to female rabbits, resulted in significant increases in haemoglobin concentration, erythrocyte volume fraction and leucocyte number in blood, as well as plasma total proteins, and urea. The present study was designed to investigate whether the hydrolytic products of MIC, methylamine (MA) and N,N'-dimethylurea (DMU) play any role in eliciting these changes. Both MA and DMU administered subcutaneously in an equimolar dose to that of 1.0 LD50 MIC, 2.2 mmol kg-1, had no influence on these parameters, although there was a marginal increase in the plasma urea level shortly after the administration of DMU. This study establishes that the observed haematological and biochemical changes induced by MIC intoxication in rabbits are mostly due to MIC.

Influence of methylamine and N,N'-dimethylurea, the hydrolysis products of methyl isocyanate, on its systemic toxicity

Subcutaneous administration of the LD50 dose of methyl isocyanate (MIC) to rats induced severe hyperglycaemia, lactic acidosis and uraemia in rats. Neither methylamine (MA) nor N,N′-dimethylurea (DMU), the hydrolysis products of MIC, administered in equimolar doses had any influence on these parameters except for a marginal transient increase in plasma urea by DMU. Methyl isocyanate administration led to haemoconcentration, resulting in an increase in the plasma concentration of total proteins and a decrease in both the plasma concentration of albumin and the plasma cholinesterase activity. The hydrolysis products of MIC had no influence on any of these parameters. Thus, it seems reasonable to suggest that the systemic effects of MIC are caused by MIC per se, in spite of its high hydrolytic instability.

Reactions of palladium chloride with amino spirocyclic cyclotriphosphazenes: Isolation and Crystal Structures of Novel Mono- and Bimetallic Complexes Formed by the Hydrolysis of a .lambda.5-Diazaphospholane Ring

The reaction of the amino spirocyclic cyclotriphosphazene N3P3(NMe2)4(NHCH2CH2CH2NH) (2) with palladium chloride gives the stable chelate complex [PdCl2.2] (4). An X-ray crystallographic study reveals that one of the nitrogen atoms of the diaminoalkane moiety and an adjacent phosphazene ring nitrogen atom are bonded to the metal. An analogous reaction with the phosphazene N3P3(NMe2)4(NHCH2CH2NH) (1) gives initially a similar complex which undergoes facile hydrolysis to give the novel monometallic and bimetallic complexes [PdCl2.HN3P3(O)(NMe2)4(NHCH2CH2NH2)] (5) and [PdCl{N3P3(NMe2)4(NCH2CH2NH2)}]2(O) (6), which have been structurally characterized; in the former, an (oxophosphazadienyl)ethylenediamine is chelated to the metal whereas, in the latter, an oxobridged bis(cyclotriphosphazene) acts as a hexadentate nitrogen donor ligand in its dianionic form. Crystal data for 4 : a = 14.137(1) angstrom, b = 8.3332(5) angstrom, c = 19.205(2) angstrom, beta = 96.108(7)degrees, P2(1)/c, Z = 4, R = 0.027 with 3090 reflections (F > 5sigma(F)). Crystal data for 5 : a = 8.368(2) angstrom, b = 16.841(4) A, c = 16.092(5) angstrom, beta = 98.31(2)degrees, P2(1)/n, Z = 4, R = 0.049 with 3519 reflections (F > 5sigma(F)). Crystal data for 6 : a = 22.455(6) angstrom, b = 14.882(3) angstrom, c = 13.026(5) angstrom, 6 = 98.55(2)degrees, C2/c, Z = 4, R = 0.038 with 3023 reflections (F > 5sigma(F)).

ATP hydrolysis is required for DNA cleavage by EcoPI restriction enzyme

The type III restriction endonuclease EcoPI, coded by bacteriophage Fl, cleaves unmodified DNA in the presence of ATP and magnesium ions. We show that purified EcoPI restriction enzyme fails to cleave DNA in the presence of non-hydrolyzable ATP analogs. More importantly, this study demonstrates that EcoPI restriction enzyme has an inherent ATPase activity, and ATP hydrolysis is necessary for DNA cleavage. Furthermore, we show that the progress curve of the reaction with Eco PI restriction enzyme exhibits a lag which is dependent on the enzyme concentration. Kinetic analysis of the progress curves of the reaction suggest slow transitions that can occur during the reaction, characteristic of hysteretic enzymes. The role of ATP in the cleavage mechanism of type III restriction enzymes is discussed.

Role of Water in the Enzymatic Catalysis: Study of ATP plus AMP -> 2ADP Conversion by Adenylate Kinase

The catalytic conversion ATP + AMP -> 2ADP by the enzyme adenylate kinase (ADK) involves the binding of one ATP. molecule to the LID domain and one AMP molecule to the NMP domain. The latter is followed by a. phosphate transfer and then the release of two ADP molecules. We have computed a novel two-dimensional configurational free energy surface (2DCFES), with one reaction coordinate each for the LID and the NMP domain motions, while considering explicit water interactions. Our computed 2DCFES clearly reveals the existence of a stable half-open half-closed (HOHC) intermediate stale of the enzyme. Cycling of the enzyme through the HOHC state reduces the conformational free energy barrier for. the reaction by about 20 kJ/mol. We find that the stability of the HOHC state (missed in all earlier studies with implicit solvent model) is largely because of the increase of specific interactions of the polar amino acid side chains with water, particularly with the arginine and the histidine residues. Free energy surface of the LID domain is rather rugged, which can conveniently slow down LID's conformational motion, thus facilitating a new substrate capture after the product release in the catalytic cycle.

Effect of beta-lactamase-catalyzed hydrolysis of cephalosporins on peroxynitrite-mediated nitration of serum albumin and cytochrome c

The hydrolysis of beta-lactam antibiotics by beta-lactamases is one of the major bacterial defense systems. These enzymes generally hydrolyze a variety of antibiotics including the latest generation of cephalosporins, cephamycins and imipenem. In this paper, the effect of cephalosporins-based antibiotics on the peroxynitrite-mediated nitration of protein tyrosine is described. Although some of the antibiotics have weak inhibitory effect on the nitration reactions in the absence of beta-lactamase, they exhibit very strong inhibition in the presence of beta-lactamase. This is due to the elimination of heterocyclic thiol/thione moieties from cephalosporins by beta-lactamase-mediated hydrolysis. After the elimination, the thiols/thiones effectively scavenge peroxynitrite, leading to the inhibition of the nitration reactions.

Intramolecular Reactivity - Keto Participation in the Hydrolysis of N-Methyl-N-(9-Oxobicyclo[3.3.1]Nonan-2-Alpha-YL) Morpholinium Iodide, and A Cannizzaro-Type Reaction of 3-(2-Oxocyclohexyl)Propanal

Mechanistic studies of two intramolecular processes, nucleophilic displacement of N-methylmorpholinium in N-methyl-N-{9-oxobicyclo[3,3,1]nonan-2 alpha-yl}morpholinium iodide, anchimerically assisted by keto carbonyl, and a Cannizzaro-type reaction of 3-(2-oxocyclohexyl)propanal, occurring via axial hydride transfer onto the cyclohexanone, are reported.

Enzymatic resolution of dioxygenated dicyclopentadienes: Enantiopure hydrindanes, hydroisoquinolones, diquinanes and application to a synthesis of (+)-coronafacic acid

A 5, 10-dioxygenated-tricyclo[5.2.1.0(2,6)]decane derivative 6 has yielded to efficient enzymatic resolution to provide a range of chiral building blocks, whose absolute configuration has been determined through a total synthesis of naturally occuring (+)-coronafacic acid. (C) 1999 Elsevier Science Ltd. All rights reserved.

Enzymatic synthesis of flavors in supercritical carbon dioxide

Commercially important flavor esters of isoamyl alcohol, catalyzed by crude hog pancreas lipase (HPL), were synthesized under solvent-free conditions and in supercritical carbon dioxide. The esters synthesized were isoamyl acetate, isoamyl propionate, isoamyl butyrate, and isoamyl octanoate. Very low yields (3-4%) of isoamyl acetate were obtained, but high yields for the other three esters were obtained under both supercritical and solvent-free conditions. The yields of esters of the even-carbon acids, isoamyl acetate, butyrate, and octanoate, increased with increasing chain length, whereas the yield of isoamyl propionate was higher than that of isoamyl butyrate. The optimum temperature of the reaction was higher under supercritical conditions (45 degreesC) than under solvent-free conditions (35-40 degreesC). The effects of other parameters such as alcohol concentration, water concentration, and enzyme loading were investigated. An increase in the water concentration decreased the conversion significantly in supercritical carbon dioxide but not under solvent-free conditions. The optimum ratio of alcohol to acid was dependent on the extent of inhibition by the acid. Although providing a higher apparent yield by being run in a highly concentrated medium, the overall conversion under solvent-free conditions was lower than that under supercritical conditions for similar enzyme concentrations, indicating that the synthesis of esters in supercritical carbon dioxide might be a viable option.