Kinetics in the pre-steady state of the formation of cystines in ribonucleoside diphosphate reductase: evidence for an asymmetric complex

Two folded polypeptides, designated R1 and R2, respectively, combine in an as yet undefined stoichiometry to form ribonucleoside diphosphate reductase (ribonucleotide reductase) from Escherichia coli. Two pairs of cysteines in each R1 protomer have been implicated in the enzymatic mechanism. One pair, cysteines 225 and 462, is located in the active site of the enzyme and forms a cystine concomitant with the reduction of the ribonucleotide. The other pair, cysteines 754 and 759, is located near the carboxy terminus and is thought to reduce the cystine in the active site by disulfide interchange; either thioredoxin or glutaredoxin is then thought to reduce the cystine that results. Rapid quenching and site-directed immunochemistry have been used to follow the formation of the cystine in the active site and the peripheral cystine simultaneously during the pre-steady state. Prereduced R1 dimer of ribonucleoside diphosphate reductase, in the presence of ATP and CDP, was mixed with R2 dimer in an apparatus for quench flow. The reaction was quenched with a solution of acetic acid and N-ethylmaleimide, the protein was then precipitated with trichloroacetic acid, and the precipitate was separated into two portions. The percent of the cystine in the active site in one of the portions was determined as described previously [Erickson, H. K. (2000) Biochemistry 39, 9241-9250]. A similar method was employed to determine the percent of the peripheral cystine in the other portion of the precipitate. It was found that while the formation of both of these cystines was initiated by the addition of R2 dimer, presumably as products of the reduction of CDP, the peripheral cystine appeared to form more rapidly and in a higher yield than the cystine in the active site. These results demonstrate that the formation of the cystine between cysteines 754 and 759 of ribonucleotide reductase from E. coli is kinetically competent. A mechanism consistent with the prior formation of the cystine between cysteine 225 and cystene 462 as well as the kinetics for the formation of each cystine with time is presented. Because twice as much of the peripheral cystine than cystine in the active site had formed during the pre-steady state, it follows that the enzymatically competent complex between R1 dimers and R2 dimers cannot be symmetric.     

Real-time measurements of dark substrate catalysis

We have developed a novel procedure to monitor the real-time cleavage of natural unmodified peptides (dark substrates). In the competition-based assay, the initial cleavage rate of a fluorogenic peptide substrate is measured in the presence of a second substrate that is not required to exhibit any optical property change upon cleavage. Using a unique experimental design and steady-state enzyme kinetics for a two-substrate system, we were able to determine both Km and k(cat) values for cleavage of the dark substrate. The method was applied to HIV-1 protease and to the V82F/I84V drug resistant mutant enzyme. Using two different substrates, we showed that the kinetic parameters derived from the competition assay are in good agreement with those determined independently using standard direct assay. This method can be applied to other enzyme systems as long as they have one substrate for which catalysis can be conveniently monitored in real time.     

Kinetics of Cdc42 membrane extraction by Rho-GDI monitored by real-time fluorescence resonance energy transfer

The mechanisms underlying the ability of the Rho-GDP dissociation inhibitor (RhoGDI) to elicit the release of Rho-related GTP-binding proteins from membranes is currently unknown. In this report, we have set out to address this issue by using fluorescence resonance energy transfer approaches to examine the functional interactions of the RhoGDI with membrane-associated Cdc42. Two fluorescence assays were developed to monitor the interactions between these proteins in real time. The first involved measurements of resonance energy transfer between N-methylanthraniloyl GDP (MantGDP) bound to Cdc42 and fluorescein maleimide covalently attached to cysteine 79 of RhoGDI (RhoGDI-FM). This assay allowed us to directly monitor the binding of RhoGDI to membrane-associated Cdc42. The second fluorescence assay involved measurements of resonance energy transfer between membrane-associated Cdc42-MantGDP and hexadecyl(amino) fluorescein that was randomly inserted into the membrane bilayer. This assay enabled us to directly monitor the (GDI-induced) release of Cdc42 from membranes. Analyses of the rates of change in the fluorescence of Cdc42-MantGDP, which serves as a resonance energy transfer donor in both of these assays, as a function of RhoGDI concentration suggests a two-step mechanism to explain the ability of RhoGDI to stimulate the release of Cdc42 from membranes. Specifically, we propose that the GDI first binds rapidly to membrane-associated Cdc42 and then a slower isomerization occurs which represents the rate-limiting step for the dissociation of the Cdc42-RhoGDI complex from membranes. We propose that this slow step in the observed kinetics reflects the time-course of translocation of the geranyl-geranyl lipid tail of Cdc42 from the outer leaflet of the membrane to the isoprenyl binding site observed in the previously reported NMR structure of the Cdc42-RhoGDI complex [Gosser et al. (1997) Nature 387, 814].     

P3 cap modified Phe*-Ala series BACE inhibitors

With the aim of reducing molecular weight and adjusting log D value of BACE inhibitors to more favorable range for BBB penetration and better bioavailability, we synthesized and evaluated several series of P3 cap modified BACE inhibitors obtained via replacement of the P3NHBoc moiety as seen in 3 with other polar functional groups such as amino, hydroxyl and fluorine. Several promising inhibitors emerging from this P3 cap SAR study (e.g., 15 and 19) demonstrated good enzyme inhibitory potencies (BACE-1 IC(50) <50 nM) and whole cell activities (IC(50) approximately 1 microM).     

Structure of a novel c7-type three-heme cytochrome domain from a multidomain cytochrome c polymer

The structure of a novel c(7)-type cytochrome domain that has two bishistidine coordinated hemes and one heme with histidine, methionine coordination (where the sixth ligand is a methionine residue) was determined at 1.7 A resolution. This domain is a representative of domains that form three polymers encoded by the Geobacter sulfurreducens genome. Two of these polymers consist of four and one protein of nine c(7)-type domains with a total of 12 and 27 hemes, respectively. Four individual domains (termed A, B, C, and D) from one such multiheme cytochrome c (ORF03300) were cloned and expressed in Escherichia coli. The domain C produced diffraction quality crystals from 2.4 M sodium malonate (pH 7). The structure was solved by MAD method and refined to an R-factor of 19.5% and R-free of 21.8%. Unlike the two c(7) molecules with known structures, one from G. sulfurreducens (PpcA) and one from Desulfuromonas acetoxidans where all three hemes are bishistidine coordinated, this domain contains a heme which is coordinated by a methionine and a histidine residue. As a result, the corresponding heme could have a higher potential than the other two hemes. The apparent midpoint reduction potential, E(app), of domain C is -105 mV, 50 mV higher than that of PpcA.