Evolution of catalytic centers of antibodies by virtual screening of broad repertoire of mutants using supercomputer.

It is proposed to perform quantum mechanical/molecular dynamics calculations of chemical reactions that are planned to be catalyzed by antibodies and then conduct a virtual screening of the library of potential antibody mutants to select an optimal biocatalyst. We tested the effectiveness of this approach by the example of hydrolysis of organophosphorus toxicant paraoxon using kinetic approaches and X-ray analysis of the antibody biocatalyst designed de novo.

Three-dimensional structures of noncovalent complexes of Citrobacter freundii methionine γ-lyase with substrates.

Crystal structures of Citrobacter freundii methionine γ-lyase complexes with the substrates of γ- (L-1-amino-3-methylthiopropylphosphinic acid) and ß- (S-ethyl-L-cysteine) elimination reactions and the competitive inhibitor L-norleucine have been determined at 1.45, 1.8, and 1.63 Å resolution, respectively. All three amino acids occupy the active site of the enzyme but do not form a covalent bond with pyridoxal 5'-phosphate. Hydrophobic interactions between the active site residues and the side groups of the substrates and the inhibitor are supposed to cause noncovalent binding. Arg374 and Ser339 are involved in the binding of carboxyl groups of the substrates and the inhibitor. The hydroxyl of Tyr113 is a potential acceptor of a proton from the amino groups of the amino acids.

Design of pyridoxal 5'-phosphate dependent catalytic antibodies.

Modern approaches for developing antibodies with coenzyme-dependent activities are discussed for pyridoxal 5'-phosphate dependent transformation of amino acid as an example. A new type of antigens analogous to enzyme--substrate compounds is suggested for the production of such antibodies. Approaches for the development of pyridoxal antiidiotypic antibody using analogs of coenzyme--substrate compounds and corresponding apoenzyme complexes are reviewed.

Hydroxylamine-containing inhibitors of polyamine biosynthesis and impairment of colon cancer cell growth.

Polyamine synthesis (by the action of ornithine decarboxylase [ODC] and S-adenosylmethionine decarboxylase [SAMDC]) and polyamine content are high in colon cancer. In addition, colonic lumen is rich in polyamines synthesised by colonic microflora; for this reason, polyamine depletion in colon cancer may be a logical approach to impair growth of colon cancer cells. We evaluated highly specific and reportedly non-toxic hydroxylamine-containing inhibitors of ODC (1-aminooxy-3-aminopropane, APA) and SAMDC (S-(5'-deoxy-5'-adenosyl)-methylthioethyl-hydroxylamine, AMA) in human colon cancer cells (Caco-2 and HT-29) in culture. APA depleted ODC activity within 24 hr, more rapidly than did difluoromethylornithine. APA and AMA in combination (100 microM each) reduced ODC and SAMDC activities to undetectable levels within 24 hr and intracellular polyamines to 8-23% of control. The resulting growth arrest could be reversed only by twice as much spermidine as is physiologically present in the colonic lumen. In concentrations sufficient to deplete growth, APA and AMA were not toxic. Simultaneous treatment with APA, AMA, and 5-fluorouracil reduced colon cancer cell survival more potently than treatment with 5-fluorouracil alone. The hydroxylamine-containing ODC and SAMDC inhibitors APA and AMA are potent inhibitors of colon cancer cell proliferation and might be therapeutically promising in colon cancer.

Interaction of tyrosine phenol-lyase with phosphoroorganic analogues of substrate amino acids.

The phosphinic analogues of tyrosine and pyruvate were first demonstrated to be substrates in the reactions of elimination and synthesis catalyzed by tyrosine phenol-lyase. Kinetic parameters of the enzymatic process were determined, and the first enzymic synthesis of an aminophosphinic acid was carried out. Replacement of the planar HOOC-group by the tetrahedral (HO)(O)PH-group in the substrate slightly affected its affinity for the enzyme but substantially diminished the conversion rate. For phosphonic analogues, containing (HO)2(O)P group, the affinity to the enzyme was decreased considerably while the conversion was completely prevented. Thus, the structural parameters of the acid group are important not only for the affinity for the enzyme, but also for the formation of the catalytically competent conformation of the active site.

[Phosphinic analog of methionine inhibits growth of leucosis cell L1210 and transforms to phosphinic analog of S-adenosylmethionine]. / Fosfinovyi analog metionina tormozit rost leikoznykh kletok L1210 i prevrashchaetsia v fosfinovyi analog S-adenozilmetionina.

A phosphinic analogue of methionine bearing a phosphinic H(OH)(O)P fragment in place of the carboxyl group inhibited the growth of the L1210 cells and was intracellularly transformed to the phosphinic analogue of S-adenosylmethionine.

[New aminooxy analogs of biogenic polyamines]. / Novye aminookisianalogi biogennykh poliaminov.

A series of structural analogs of putrescine, spermidine, and spermine with the aminomethylene fragment substituted by the aminooxy group was suggested. The synthesis of the new aminooxy analogs of spermine was described. Biochemical aspects of the activity of the aminooxy analogs of polyamines were discussed in respect of their selective inhibition of normal and leukemic cells.

Succinyl phosphonate inhibits alpha-ketoglutarate oxidative decarboxylation, catalyzed by alpha-ketoglutarate dehydrogenase complexes from E. coli and pigeon breast muscle.

Effects of a set of alpha-ketoglutarate phosphoanalogues on the activity of alpha-ketoglutarate dehydrogenase (EC 1.2.4.2) complexes from E. coli and pigeon breast muscle, as well as on alpha-ketoglutarate dehydrogenase isolated from the pigeon breast muscle, have been studied. alpha-Ketoglutarate phosphoanalogues (succinyl phosphonate and its monomethyl ester) were found to be effective inhibitors of alpha-ketoglutarate oxidative decarboxylation, catalyzed by both muscle and bacterial alpha-ketoglutarate dehydrogenase complexes, as well as muscle alpha-ketoglutarate dehydrogenase. The ability of glutamate phosphoanalogues to inhibit alpha-ketoglutarate oxidative decarboxylation has been shown in E. coli extract and a model system.

Crystal structures and solution studies of oxime adducts of mitochondrial aspartate aminotransferase.

The interaction of mitochondrial aspartate aminotransferase with hydroxylamine and five derivatives (in which the hydroxyl hydrogen is replaced by the side chain of naturally occurring amino acids) was investigated by X-ray diffraction as well as by kinetic and spectral measurements with the enzyme in solution. The inhibitors react with pyridoxal 5'-phosphate in the enzyme active site, both in solution and in the crystalline state, in a reversible single-step reaction forming spectrally distinct oxime adducts. Dissociation constants determined in solution range from 10(-8) M to 10(-6) M depending on the nature of the side-chain group. The crystal structures of the adducts of mitochondrial aspartate aminotransferase with the monocarboxylic analogue of L-aspartate in the open and closed enzyme conformation were determined at 0.23-nm and 0.25-nm resolution, respectively. This inhibitor binds to both the open and closed crystal forms of the enzyme without disturbing the crystalline order. Small differences in the conformation of the cofactor pyridoxal phosphate were detected between the omega-carboxylate of the inhibitor and Arg292 of the neighbouring subunit is mainly responsible for the attainment of near-coplanarity of the aldimine bond with the pyridine ring in the oxime adducts. Studies with a fluorescent probe aimed to detect shifts in the open/closed conformational equilibrium of the enzyme in oxime complexes showed that the hydroxylamine-derived inhibitors, even those containing a carboxylate group, do not induce the 'domain closure' in solution. This is probably due to the absence of the alpha-carboxylate group in the monocarboxylic hydroxylamine-derived inhibitors, emphasizing that both carboxylates of the substrates L-Asp and L-Glu are essential for stabilizing the closed form of aspartate aminotransferase.

The reduction of thymine residues in DNA by the combined action of UV light and hypophosphite.

UV irradiation (lambda = 254 nm) of thymine and uracil in aqueous solution containing salts of phosphinic acid (hypophosphites) results in the formation of the corresponding dihydropyrimidine derivatives. The peculiarities of this new photochemical reaction consist of a specificity towards 2,4-dioxophyrimidines, a high quantum yield and a neutral pH optimum. The quantum yield of photoconversion of thymine at pH 7.0 is equal to 1.9 x 10(-2) in 1 M NaH2PO2; it is diminished to 8.5 x 10(-3) in 0.1 M NaH2PO2. The same decrease in quantum yield with concentration is also found for uracil and uridine; quantum yields of their transformations in 0.1 M NaH2PO2 at pH 7.0 are 6.6 x 10(-2) and 1.5 x 10(-1) respectively. The mild conditions and high quantum yields characteristic of the photoinduced reaction above open up the possibility of obtaining DNA molecules which contain among pyrimidine photoproducts mainly dihydropyrimidine residues. A correlation between various types of thymine modifications in UV-irradiated double-stranded DNA (dimers and dihydrothymidine residues) and the amplitude of the intense negative band in the circular dichroism (CD) spectra specific for DNA liquid crystalline dispersions has been established. A possible application of this base-specific modification to the investigation of nucleic acids is considered.

Aminooxy analogues of spermidine evidence the divergent roles of the charged amino nitrogens in the cellular physiology of spermidine.

Two recently devised spermidine analogues, N-[2-aminooxyethyl]-1,4-diaminobutane (AOEPU) and 1-aminooxy-3-N-[3-aminopropyl]-aminopropane (APAPA), were used to elucidate the role of charge distribution in the functions of spermidine in cultured baby hamster kidney cells. The drugs did not affect cell proliferation nor did they relieve the growth-arrest but potentiated the metabolic disturbances caused by DL-alpha-difluoromethyl-ornithine (DFMO). Neither drug affected spermidine uptake but both competed with putrescine uptake. Neither drug could replace spermidine in the control of S-adenosylmethionine decarboxylase and accumulation of the reaction product. APAPA prevented spermine synthesis and showed that modest putrescine synthesis take place in the presence of DFMO. AOEPU, but not APAPA, interfered with cellular constituents resulting in enzymatic formation, accumulation and excretion to culture medium of UV-absorbing catabolites.

Application of oxime formation in a radiometric assay of aminooxy compounds.

A radiometric determination of monoaminooxy analogues of naturally occurring polyamines is described in which [2-14C]acetone is employed as reagent. The reagent is volatile while the oxime product is not allowing unreacted reagent to be removed and the oxime formation to be completed by lyophilization in vacuo. The residual radioactive compound is soluble in water and proportional to the reactive aminooxy content of the reaction mixture and can be quantified by liquid scintillation counting. The assay method is inexpensive and simple and has high specificity and flexibility in sample volume enabling reliable quantification of reactive aminooxy amines in biological extracts at concentrations exceeding 0.25 microM. Optimal pH values for oxime formation of five monoaminooxy analogues of polyamines with acetone were resolved. Reactions via reversible intermediates to irreversible oximes were sped up by removal of water. Complete oxime formation and stability was confirmed by 1H NMR studies. Tested drugs readily formed oximes with pyridoxal 5'-phosphate, too. Diaminooxy analogue of cadaverine formed a volatile oxime with acetone. The method was used to monitor the stability of aminooxy analogues of putrescine and spermidine during storage and under culture conditions and to establish their scant accumulation in, but fast catabolism by, cultured baby hamster kidney cells.