Enantio-selectivity of human nucleoside monophosphate kinases

Over recent years, there has been a renewed interest in the development of L-nucleosides as safe and efficacious drugs for the treatment of viral infections. Biological activity of these compounds requires phosphorylation to their triphosphate form, involving nucleoside monophosphate kinases in the second step. In order to characterize the activation pathway of L-nucleosides of the pyrimidine series, we studied the enantio-selectivity of human uridylate-cytidylate and thymidylate kinases. The results showed that these enzymes are only weakly enantio-selective and are thus probably involved in the activation of L-nucleosides in vivo. An activation pathway for telbivudine (L-dT) was therefore proposed.status: publishe

Reaction of human UMP-CMP kinase with natural and analog substrates.

International audienceUMP-CMP kinase catalyses an important step in the phosphorylation of UTP, CTP and dCTP. It is also involved in the necessary phosphorylation by cellular kinases of nucleoside analogs used in antiviral therapies. The reactivity of human UMP-CMP kinase towards natural substrates and nucleotide analogs was reexamined. The expression of the recombinant enzyme and conditions for stability of the enzyme were improved. Substrate inhibition was observed for UMP and CMP at concentrations higher than 0.2 mm, but not for dCMP. The antiviral analog l-3TCMP was found to be an efficient substrate phosphorylated into l-3TCDP by human UMP-CMP kinase. However, in the reverse reaction, the enzyme did not catalyse the addition of the third phosphate to l-3TCDP, which was rather an inhibitor. By molecular modelling, l-3TCMP was built in the active site of the enzyme from Dictyostelium. Human UMP-CMP kinase has a relaxed enantiospecificity for the nucleoside monophosphate acceptor site, but it is restricted to d-nucleotides at the donor site

Steady‐state kinetics and analysis of pH dependence on wild‐type and a modified allosteric Pseudomonas aeruginosa ornithine carbamoyltransferase containing the replacement of glutamate 105 by alanine

The substitution of alanine for glutamate at position 105 (E105 A) of the allosteric ornithine carbamoyltransferase (OTCase) of Pseudomonas aeruginosa abolishes the carbamoylphosphate (CP) cooperativity observed in the wild‐type enzyme. A kinetic analysis of [E105 A]OTCase was performed in order to determine the mechanism of the reaction. The results of initial velocity and inhibition studies are consistent with an ordered mechanism with CP as the first substrate to add to the enzyme. In addition, similar studies have been made using the wild‐type enzyme in the presence of the activator, phosphate. The results are similar to those obtained with [E105 A]OTCase indicating that the residue E105 is critical for the allosteric transition of the wild‐type enzyme. The activities of the wild‐type allosteric OTCase and of [E105 A]OTCase have been studied in the pH range 5.8–8.2 in the absence and in the presence of positive and negative effectors. The sigmoid saturation of OTCases by CP has been analyzed according to the Hill equation. At low pH values, CP cooperativity is low in the wild‐type enzyme but cooperativity and [S]CP 0.5 values increase markedly with pH. For [E105 A]OTCase, the saturation by CP is hyperbolic at all pH values; in this modified enzyme, the presence of spermidine, an allosteric inhibitor of the wild‐type enzyme, results in an inhibition which induces CP cooperativity. Thus, the ionization of the residue E105 apparently results in a conformational change in the wild‐type enzyme which modifies the catalytic site. Since the [E105 A]enzyme retains the heterotropic effects of the wild‐type enzyme, other structural features are required for the allosteric transition in the wild‐type catabolic OTCase. Copyright © 1993, Wiley Blackwell. All rights reservedSCOPUS: ar.jinfo:eu-repo/semantics/publishe

Adenosine phosphonoacetic acid is slowly metabolized by NDP kinase

NDP kinase catalyzes the last step in the phosphorylation of nucleotides. It is also involved in the activation by cellular kinases of nucleoside analogs used in antiviral therapies. Adenosine phosphonoacetic acid, a close analog of ADP already proposed as an inhibitor of ribonucleotide reductase, was found to be a poor substrate for human NDP kinase, as well as a weak inhibitor with an equil. dissocn. const. of 0.6 mM to be compared to 0.025 mM for ADP. The X-ray structure of a complex of adenosine phosphonoacetic acid and the NDP kinase from Dictyostelium was detd. to 2.0 A resoln. showing that the analog adopts a binding mode similar to ADP, but that no magnesium ion is present at the active site. As ACP may also interfere with other cellular kinases, its potential as a drug targeting NDP kinase or ribonucleotide reductase is likely to be limited due to strong side effects. The design of new mols. with a narrower specificity and a stronger affinity will benefit from the detailed knowledge of the complex ACP-NDP kinas

Acyclic phosphonate nucleotides and human adenylate kinases: impact of a borano group on alpha-P position

Adenylate kinases are involved in the activation of antiviral drugs such as the acyclic phosphonates analogs PMEA and (R)PMPA. We examine the in vitro phosphorylation of PMEA and PMPA bearing a borano- or a H- group on the phosphorus atom. The alpha-borano or alpha-H on PMEA and PMPA were detrimental to the activity of recombinant human AMP kinases 1 and 2. Docking PMEA to the active site of AMP kinase 1 indicated that the borano group may prevent two conserved critical Arg interactions with the alpha-phosphate, resulting in substrate bad positioning.status: publishe