Elena N. Khurs

Fhit proteins can also recognize substrates other than dinucleoside polyphosphates

We show here that Fhit proteins, in addition to their function as dinucleoside triphosphate hydrolases, act similarly to adenylylsulfatases and nucleoside phosphoramidases, liberating nucleoside 5′‐monophosphates from such natural metabolites as adenosine 5′‐phosphosulfate and adenosine 5′‐phosphoramidate. Moreover, Fhits recognize synthetic nucleotides, such as adenosine 5′‐O‐phosphorofluoridate and adenosine 5′‐O‐(γ‐fluorotriphosphate), and release AMP from them. With respect to the former, Fhits behave like a phosphodiesterase I concomitant with cleavage of the P–F bond. Some kinetic parameters and implications of the novel reactions catalyzed by the human and plant (Arabidopsis thaliana) Fhit proteins are presented.

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

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

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.

Methionine γ-lyase: Mechanistic deductions from the kinetic pH-effects: The role of the ionic state of a substrate in the enzymatic activity

We have studied and compared the pH-dependencies of the main kinetic parameters for the alpha,gamma-elimination reactions of methionine gamma-lyase (MGL) of Citrobacter intermedius with natural substrate, l-methionine, with its phosphinic analogue, and for alpha,beta-elimination reaction with S-methyl-l-cysteine. From the pH-dependency of k(cat)/K(m) for the reaction with l-methionine we have concluded that MGL is selective with respect to the zwitterionic form of its natural substrate. For the reaction of MGL with 1-amino-3-methylthiopropylphosphinic acid the pK(a) of the substrates amino group, equal to 7.55, is not reflected in the pH-profile of k(cat)/K(m). Consequently, the enzyme does not manifest well-defined selectivity with respect to the zwitterion and anion ionic forms of the substrate. The ascending limbs of pH-dependencies of k(cat)/K(m) for reactions with l-methionine and S-methyl-l-cysteine are controlled by a single pK(a) equal to 7.1-7.2, while for the reaction with 1-amino-3-methylthiopropylphosphinic acid two equal pK(a)s of 6.2 were found in the respective pH-profile. The descending limbs of pH-dependencies of k(cat)/K(m) for the reactions with S-methyl-l-cysteine and racemic 1-amino-3-methylthiopropylphosphinic acid are very similar and are controlled by two acidic groups having average pK(a) values of 8.7. On the basis of these results we suggest a mechanism of catalytic action of MGL. According to this mechanism Tyr 113, in its conjugated base form, acts as an acceptor of the proton from the amino group of the substrate upon its binding in the active site. Elimination of the leaving thiol groups during both alpha,gamma- and alpha,beta-elimination reactions is assisted by the acidic groups of Tyr 113 and Tyr 58. Both tyrosyl residues are able to fulfill this catalytic function with different efficiencies depending on the type of elimination reaction. Tyr 113 residue plays the determining role in the alpha,gamma-elimination, and Tyr 58 - in the alpha,beta-elimination process.

Synthesis of (α-substituted α-aminophosphinic and α-aminophosphonic acids

The reaction of ketoximes with hypophosphorous acid resulted in previously unknown α-substituted-α-aminophosphinic acids, which were oxidized into the corresponding α-substituted-α-aminophosphonic acids.

Synthesis of phosphinic and phosphoric analogs of aspartic acid

Approaches to the synthesis of 1-amino- and 2-amino-2-carboxyethylphosphinic and-phosphoric acids have been studied. A convenient method for the preparation of phosphinic acids is the reactions of ethyl diethoxymethylphosphonite with ethyl acetamidomethylenemalonate and ethyl 2-acetamidoacrylate.

Aminoalkythiophosphonates: a new type of biologically active compounds.

The broad family of natural and synthetic organophosphorous analogues of amino acids includes a large group of compounds classified with phosphinic 1 and phosphonic 2 acids (scheme 1 ), whose diverse physiological activity is determined by their effect on amino acid exchange and depends on the structure of the phosphorus-containing fragment. For example, substitution of the monobasic carboxyl group of an amino acid with the monobasic P(O)(OH)H group does not qualitatively affect the affinity of aminoalkylphosphinates 1 for enzymes involved in amino acid exchange and the ability of these isosteres of amino acids for substrate transformations, as well as does not prevent their permeation across microbial cell walls [1]. In aminoalkylphosphonates 2 , the dibasic phosphonate group significantly differs in size, molecular geometry, and acidity from the carboxyl group of amino acids, making the concept of the direct structural similarity between phosphonates 2 and amino acids fairly formal [2]. However, the structure of phosphonates 2 allows them to be considered as stable analogues of labile transient states or intermediate compounds in enzymatic transformations of amino acids [3, 4]. This led to occurrence of novel inhibitors of enzymes involved in amino acid exchange and haptens for designing catalytic antibodies. Thus, modification of the phosphorus-containing fragment may induce changes in the basic biochemical characteristics of phosphorous analogues, as is observed in the case of phosphinates 1 and phosphonates 2 .

Synthesis of phosphinic and phosphonic analogs of homoserine

A convenient procedure for the synthesis of 1-amino-3-hydroxypropylphosphinic and -phosphonic acids (analogs of homoserine) was developed. The procedure involves the reaction of salts of phosphinic and phosphonic analogs of S-methylmethionine with AcONa/AcOH followed by hydrolysis.

Aminoalkylphosphinates Are New Effective Inhibitors of Melanogenesis and Fungicides

Phytopathogenic fungi exhibit high resistance to extremal environmental conditions, which is largely determined by protective properties of high-molecularweight pigments of different composition contained in them. Melanin of cell walls of the pathogen Magnaporthe grisea that causes blast disease of rise is classified in the widespread type of pigments; it represents a colored polymer of 1,8-dihydroxynaphthalene, the biosynthesis of which depends on acetyl-CoA (the pentaketide pathway) [1]. Analysis of the strains of this fungus defective in pigmentation, which were obtained by induced mutagenesis, showed that blockade of the biosynthesis of melanin attenuates the ability of the fungus to penetrate into leaf tissues and prevents excrescence of the mycelium in the plant, thereby suppressing its pathogenicity [2]. One of the most effective preparations that are used for fighting against blast disease of rice, tricyclazole 1 (Scheme 1), is classified with a small group of fungicides of similar structure, the effect of which is related to selective blockade of the synthesis of the pigment [1]. Another type of inhibitors, which has been represented so far only by the organophosphorous analogue of alanine 2.1 , suppresses the biosynthesis of not only melanin but also other metabolites of the polyketide pathway (including phytotoxins) and exhibits fungitoxicity [3]. In contrast to tricyclazole, which is effective per se , analogue 2.1 is the precursor of the active compound, pyruvate analogue 3.1 , which suppresses the synthesis of acetyl-CoA and melanin (Scheme 2) [4]. A similar mechanism was suggested for the blockade with analogue 2.1 of formation of plant polyketide pigment [5].

Fungicidal Activity of Phosphinic Analogues of Amino Acids Involved in Methionine Metabolism

In medicine and agriculture, the necessity of rationally planning attempts to find for new biologically active chemical compounds because of a tendency towards a reduction in the rate of the introduction of new products [1]. The conventional approach, based on accidental discovery of new preparations with their subsequent chemical optimization, has proven to be inefficient. With respect to pesticides, it implies synthesis and testing of 10000–15000 compounds in order to develop a single valuable preparation, i.e., this approach is time-consuming and expensive. Using the example of a new kind of fungicide, we have shown in this study that a rational search for biologically active compounds may be based on a system of chemical regulators of certain metabolic pathways. This new approach is much more efficient.