Mutation of one of the cysteine residues in the redox active disulfide of thioredoxin reductase from Escherichia coli results in C135S with CysI3' remaining or C138S with CysI3' remaining. The expression system for the genes encoding thioredoxin reductase, wild-type enzyme, C135S, and C138S has been re-engineered to allow for greater yields of protein. Wild-type enzyme and C135S were found to be as previously reported, whereas discrepancies were detected in the characteristics of C138S. It was shown that the original C138S was a heterogeneous mixture containing C138S and wild-type enzyme and that enzyme obtained from the new expression system is the correct species. C138S obtained from the new expression system having 0.1 % activity and 7% flavin fluorescence of wild-type enzyme was used in this study. Reductive titrations show that, as expected, only 1 mol of sodium dithionite/mol of FAD is required to reduce C138S. The remaining thiol in C135S and C138S has been reacted with 5,5'-dithiobis-(2-nitrobenzoic acid) to form mixed disulfides. The half time of the reaction was <5 s for in C135S and approximately 300 s for C Y S '~~ in (2138s showing that CysI3' is much more reactive. The resulting mixed disulfides have been reacted with Cys32 in C35S mutant thioredoxin to form stable, covalent adducts C138S-C35S and C135S-C35S. The half times show that Cys"' is approximately fourfold more susceptible to attack by the nucleophile. These results suggest that Cys 13' may be the thiol initiating dithiol-disulfide interchange between thioredoxin reductase and thioredoxin.Keywords: dithiol-disulfide interchange; flavoprotein; thioredoxin reductase Thioredoxin reductase from Escherichia coli is a pyridine nucleotidedisulfide oxidoreductase containing FAD and a disulfide in each active site. The redox active disulfide is composed of C Y S "~ and Cys'". Reducing equivalents move from NADPH to FAD, from Abbreviations: DTNB, 5,5'-dithiobis-(2-nitrobenzoic acid); TNB anion, 5-thio-2-nitrobenzoate anion: DTT, 1,4-dithiothreitol; DPDS, 4,4'-dithio-C138S and C135S, thioredoxin reductase in which C Y S '~~ or C Y S '~~ has dipyridine: PDS, 4-thiopyridone; IPTG, isopropyl P-D-thiogalactoside; been changed to Ser, respectively; C32S and C35S, thioredoxin in which Cys32 or has been changed to Ser, respectively: C138S-C35S, a covalent adduct where the remaining active site thiol of C138S is linked via a disulfide to the remaining active site thiol of C35S, analogous designations are used for the three other possible combinations of mutated thioredoxin reductases and thioredoxins. reduced flavin to the active site disulfide; dithiol-disulfide interchange effects reduction of the substrate thioredoxin. The redox active disulfide of thioredoxin is composed of Cys3' and Cys3'. Modification by site-directed mutagenesis of the active site in the enzyme has produced the single thiol mutants C138S (with CysI3' remaining from the active site disulfide) and C135S (with CysI3' remaining) (Prongay et al., 1989). The spectral characteristics...

Common variation in the CYP 2B6 gene, encoding the cytochrome P450 2B6 enzyme, is associated with substrate‐specific altered clearance of multiple drugs. CYP 2B6 is a minor contributor to hepatic nicotine metabolism, but the enzyme has been proposed as relevant to nicotine‐related behaviors because of reported CYP 2B6 mRNA expression in human brain tissue. Therefore, we hypothesized that CYP 2B6 variants would be associated with altered nicotine oxidation, and that nicotine metabolism by CYP 2B6 would be detected in human brain microsomes. We generated recombinant enzymes in insect cells corresponding to nine common CYP 2B6 haplotypes and demonstrate genetically determined differences in nicotine oxidation to nicotine iminium ion and nornicotine for both (S) and (R)‐nicotine. Notably, the CYP 2B6.6 and CYP 2B6.9 variants demonstrated lower intrinsic clearance relative to the reference enzyme, CYP 2B6.1. In the presence of human brain microsomes, along with nicotine‐ N ‐oxidation, we also detect nicotine oxidation to nicotine iminium ion. However, unlike N ‐oxidation, this activity is NADPH independent, does not follow Michaelis‐Menten kinetics, and is not inhibited by NADP or carbon monoxide. Furthermore, metabolism of common CYP 2B6 probe substrates, methadone and ketamine, is not detected in the presence of brain microsomes. We conclude that CYP 2B6 metabolizes nicotine stereoselectively and common CYP 2B6 variants differ in nicotine metabolism activity, but did not find evidence of CYP 2B6 activity in human brain.

All known guanidino kinases contain a conserved cysteine residue that interacts with the nonnucleophilic η 1 -nitrogen of the guanidino substrate. Site-directed mutagenesis studies have shown that this cysteine is important, but not essential for activity. In human muscle creatine kinase (HMCK) this residue, Cys283, forms part of a conserved cysteine-proline-serine (CPS) motif and has a pK a about 3 pH units below that of a regular cysteine residue. Here we employ a computational approach to predict the contribution of residues in this motif to the unusually low cysteine pK a . We calculate that hydrogen bonds to the hydroxyl and to the backbone amide of Ser285 would both contribute ~1 pH unit, while the presence of Pro284 in the motif lowers the pK a of Cys283 by a further 1.2 pH units. Using UV difference spectroscopy the pK a of the active site cysteine in WT HMCK and in the P284A, S285A and C283S/S285C mutants was determined experimentally. The pK a values, although consistently about 0.5 pH units lower, were in broad agreement with those predicted. The effect of each of these mutations on the pH-rate profile was also examined. The results show conclusively that, contrary to a previous report Biochemistry 40, 11698-11705), Cys283 is NOT responsible for the pK a of 5.4 observed in the WT V/K creatine pH profile. Finally we use molecular dynamics simulations to demonstrate that, in order to maintain the linear alignment necessary for associative inline transfer of a phosphoryl group, Cys283 needs to be ionized.Creatine kinase (CK, E.C. 2.7.3.2) catalyzes the reversible transfer of the γ-phosphoryl group of ATP to creatine (Cr), forming ADP and phosphocreatine (PCr). The latter is considered to be a reservoir of "high-energy phosphate" which is able to supply ATP, the primary energy source in bioenergetics, on demand. As a result, CK plays a major role in energy homeostasis of cells with intermittently high energy requirements, such as skeletal and cardiac muscle, neurons, photoreceptors, spermatozoa and electrocytes (1-3). CK is found in all vertebrates and exists in a variety of isoforms including the muscle, brain and mitochondrial isozymes. The two mitochondrial isozymes, ubiquitous (Mi u ) and sarcomeric (Mi s ), can exist as dimers but are generally octameric. The subunits of the muscle (M) and brain (B) isozymes, each with a molecular mass of ~43 kDa, form homodimers (MM, BB). In addition, they form a heterodimer (MB) which is used as a marker for myocardial infarction (4,5). In fact, cellular CK levels are perturbed in a number of human disease states including neurodegenerative diseases (6,7), muscular dystrophies (8) and cancer (9-11).