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Results show that the activity restoration in the mutant thymidylate synthase follows a sequential pattern of subunit separation and their subsequent recombination to an active heterodimer. Sequence data of the homo- and heterodimers of the enzyme indicate that the active enzyme is a product of subunit exchange.

A study was conducted to analyze the crystal structures of rat thymidylate synthase (TS) complexed with dUMP and the anticancer drug Tomudex. Recombinant rat hepatoma TS was determined from an overexpressing Escherichia coli. Experimental results indicated that the rat protein undergoes a ligand-induced conformation change. Findings also showed an open conformation in the structures that may be correlated with the accumulation of various destabilizing factors.

The pteridine reductase 1 of Leishmania major catalyzes the pterin and folate metabolism in this protozoan parasite. The enzyme renders Leishmania resistant to the action of methotrexate. Stereochemical and kinetic analyses were undertaken to further characterize this enzyme and assign it to a particular enzyme group. Results indicate that pteridine reductase belongs to the family of short chain dehydrogenases.

The catalysis done by two enzymes is electrostatically guided by glutamic acid. Reduced rate of bond cleavage of the folyl N5-methylene bond is due to the loss of electrostatic destabilization by the side-chain carboxylate of Glu 58/60 of the anionic intermediates resulting from deprotonation. Replacement of Glu 58 in thymidylate synthase(TS) and Glu-60 in dCMP hydroxymethylase (CH) by site-directed mutagenesis separates initial steps in catalysis by TS and CH from following steps. They have accessory roles in initial nucleotide binding.

Analysis of the influence of alpha-secondary tritium isotope on deoxycytidylate (dCMP) hydroxymethylase (CH)-catalyzed reactions reveals that CH-induced catalysis occurs through sequential hybridization modifications at C6 position of the nucleotides. The nucleotide analog 5-fluorodeoxuridylate (fdUMP) forms a covalent complex to inactivate CH. The equilibrium isotopic and kinetic isotopic effects have values of 0.8 and 1.0, respectively, and constitute the variations in the relative rates of catalysis in dUMP and dCMP turnovers.

Experimental studies with T4 lysozyme and various other proteins reveal the importance of genetic techniques in the investigation of structure-activity relationship of enzymes. Genetic techniques help determine the correlation between amino acid sequence and protein structure and understand the enzyme's catalytic mechanisms. Application of site-directed mutagenesis to examine the hypothesis derived from considering the protein's structure is a powerful and efficient method.

Catalysis of O-18 incorporation from solvent water into the final intermediate product is caused by the bacteriophage T4 deoxycytidylate (dCMP) hydroxymethylase. The final intermediate product, HmdCMP, is identified by the O-18 exchange experiments designed for identification. The catalysis occurs in dCMP in the presence of tetrahydrofolate and produces a resynthesis of covalent enzymes by a reverse reaction.

Site-directed mutagenesis was used to study the roles played by Cys148 and Asp179 in catalysis by deoxycytidylate (dCMP) hydroxymethylase (CH). Results showing that converting Cys148 tp Asp, Gly or Ser causes CH activity to decline 10(super 5)-fold suggests that Cys148 plays anucleophilic role in the process. Asp179 conversion to Asn causes a 1.5 x 10(super 4)-fold decrease in the dCMP kcat/Km value and a 60-fold rise in the dUMP kcat/Km value. Results also suggest that Asp179 stabilizes covalent catalytic intermediates by protonating the pyrimidine-CH adduct's N3.

Pteridine reductase (PTR1) is a short-chain reductase (SDR) responsible for the salvage of pterins in parasitic trypanosomatids. PTR1 catalyzes the NADPH-dependent two-step reduction of oxidized pterins to the active tetrahydro-forms and reduces susceptibility to antifolates by alleviating dihydrofolate reductase (DHFR) inhibition. Crystal structures of PTR1 complexed with cofactor and 7,8-dihydrobiopterin (DHB) or methotrexate (MTX) delineate the enzyme mechanism, broad spectrum of activity and inhibition by substrate or an antifolate. PTR1 applies two distinct reductive mechanisms to substrates bound in one orientation. The first reduction uses the generic SDR mechanism, whereas the second shares similarities with the mechanism proposed for DHFR. Both DHB and MTX form extensive hydrogen bonding networks with NADP(H) but differ in the orientation of the pteridine. [ABSTRACT FROM AUTHOR]

Mutations that suppress the defects introduced into T4 lysozyme by single amino acid substitutions were isolated and characterized. Among 53 primary sites surveyed, 8 yielded second-site revertants; a total of 18 different mutants were obtained. Most of the restorative mutations exerted global effects, generally increasing lysozyme function in a number of primary mutant contexts. Six of them were more specific, suppressing only certain specific deleterious primary substitutions, or diminishing the function of lysozymes bearing otherwise nondeleterious primary substitutions. Some variants of proteins bearing primary substitutions at the positions of Asp 20 and Ala 98 are inferred to have significantly altered structures. [ABSTRACT FROM AUTHOR]

The present work expands the chemical space known to offer potent inhibition of the serotonin transporter (SERT), norepinephrine transporter (NET), and dopamine transporter (DAT) and discloses novel bicyclic octahydrocyclopenta[c]pyrrole and octahydro-1H-isoindole scaffolds as potent triple reuptake inhibitors (TRIs) for the potential treatment of depression. Optimized compounds 22a (SERT, NET, DAT, IC(50) = 20, 109, 430 nM), 23a (SERT, NET, DAT, IC(50) = 29, 85, 168 nM), and 26a (SERT, NET, DAT, IC(50) = 53, 150, 140 nM) were highly brain penetrant, active in vivo in the mouse tail suspension test at 10 and 30 mpk PO, and were not generally motor stimulants at doses ranging from 1 to 30 mpk PO. Moderate in vitro cytochrome P450 (CYP) and potassium ion channel Kv11.1 (hERG) inhibition were uncovered as potential liabilities for the chemical series.