Substrate and cofactor binding to fluorescently labeled cytoplasmic malate dehydrogenase.

Cytoplasmic malate dehydrogenase (cMDH) is a key enzyme in several metabolic pathways. Though its activity has been examined extensively, there are lingering mechanistic uncertainties involving substrate and cofactor binding. To more completely understand this enzyme's interactions with cofactor and substrate ligands, a fluorescent reporter group was introduced into the enzyme's structure. This was accomplished by selective modification of Cys 110. The reaction placed an aminonaphthaline sulfonic acid group near the enzyme's active site. Substrate, inhibitor, and NAD binding activities were characterized using changes in this label's fluorescence. Results demonstrated that both substrate and cofactor molecules bound to the enzyme in the absence of their companion ligands. This is in contrast to strictly ordered cofactor then substrate binding as has been suggested by kinetic analyses of closely related enzymes. Binding results also indicated that the cofactor, NAD, bound to cMDH in a negatively cooperative manner, but substrates and the inhibitor, hydroxymalonate, bound non-cooperatively. Multiple substrate binding modes were identified and interactions between substrate and cofactor binding were found.

Characterization and use of a recombinant retroviral system for the analysis of drug resistant HIV.

A recombinant retroviral system was used for the analysis of early HIV breakthrough infection in the presence of antiviral drugs. The use of replication-defective HIV allowed a quantitative analysis of a single cycle of infection. This report characterizes this recombinant HIV system and demonstrates it's validity in comparison to standard assays. It is demonstrated that the protease inhibitor XM323 inhibits both early and late events in the HIV life-cycle, while dextran sulphate inhibits only early events. In addition, it is shown that this system can be used for detecting and quantitating drug resistant HIV. Thus, the use of this system may provide both novel information about the stage of the viral life-cycle inhibited and a preliminary assessment of the mechanism(s) responsible for breakthrough infection in the presence of antiretroviral drugs.

3'-Amino thymidine affinity matrix for the purification of herpes simplex virus thymidine kinase.

A simple procedure for preparation of an affinity resin with 3'-amino thymidine linked to the carboxyl residues on 6-amino-hexanoic agarose is described. We have used this column for a rapid and simple purification of the thymidine kinase encoded by the herpes simplex virus type 1 genome. This resin has two major advantages over the most widely use used resin made with thymidine-p-nitrophenyl phosphate: first it is easily obtainable, and second, it is not subject to destruction by phosphodiesterases. The two resins are very similar in behavior and the resin made with amino thymidine has allowed us to prepare large quantities of highly purified HSV TK for crystallization studies.

Sanctuary growth of human immunodeficiency virus in the presence of 3'-azido-3'-deoxythymidine.

Human immunodeficiency virus (HIV) resistance to the nonnucleoside reverse transcriptase inhibitors emerges very rapidly under selection in culture and in patients. In contrast, zidovudine (3'-azido-3'-deoxythymidine [AZT])-resistant HIV generally emerges in patients only after more-prolonged therapy. Although HIV can be cultured from many patients shortly after the initiation of AZT treatment, characterization of the virus that is cultured generally indicates that it is sensitive to AZT. To initiate an evaluation of the mechanisms contributing to early HIV breakthrough in the presence of AZT and other nucleoside analogs, we have utilized replication-defective HIV encoding reporter genes. These recombinant HIV allow a quantitative analysis of a single cycle of infection. Results with these defective HIV indicate that early infection in the presence of AZT often results from the infection of a cell which is refractory to the antiretroviral effects of AZT. Characterization of a cell line derived from one such cell has demonstrated decreased accumulation of AZT triphosphate, increased phosphorylation of thymidine to thymidine triphosphate, and increased levels of thymidine kinase activity. In addition, AZT inhibition of replication-competent HIV infection is also significantly impaired in this cell line. Attempts to detect and characterize the mechanisms responsible for early viral infection after initiation of AZT therapy may result in the development of new strategies for prolonged suppression of viral infection prior to the emergence of drug-resistant virus.

Substrate specificity of Epstein-Barr virus thymidine kinase.

Purified recombinant protein encoded by the BXLF-I open reading frame of the Epstein-Barr virus genome has thymidine kinase activity. The substrate behaviors of various nucleosides toward this enzyme were tested. Halogenated deoxyuridines, zidovudine, and bromovinyldeoxyuridine are efficient substrates, while acyclovir and dihydroxypropylmethylguanine are relatively poor substrates for the Epstein-Barr virus thymidine kinase.

Ligand-induced asymmetry between active sites of cytoplasmic malate dehydrogenase: a chemical modification study.

The iodoacetate-dependent and iodoacetamide-dependent inhibition of cytoplasmic malate dehydrogenase (s-MDH) has been examined. We have confirmed previous reports that iodoacetate inhibits this dimeric enzyme by modifying a single active site methionine per s-MDH subunit. Time courses for the inactivation of the solution-state enzyme with both reagents indicate each s-MDH subunit is modified with equal rapidity in the absence of substrate or cofactor. However, the subunits react with distinctly different rates in the presence of cofactor or cofactor/substrate combinations, indicating some conformational asymmetry between subunits occurs when these ligands are bound. This is consistent with solution-state s-MDH behaving as a cooperative enzyme. Apo and holo crystalline s-MDH are also inhibited by iodoacetic acid. However, subunits of the crystalline enzyme are inhibited with different rates in the presence or absence of active site ligands. This suggests subunit conformations of the dimeric enzyme are not identical in crystalline s-MDH preparations regardless of ligand binding. Furthermore, by the criterion of inhibition rate constants, subunit conformations of the crystalline enzyme are not rigid but are perturbed by ligand binding. Comparisons of inactivation time courses for solution- and crystalline-state s-MDH suggest crystalline s-MDH exhibits at least some of the subunit asymmetry associated with the solution-state enzyme.