Ground-state destabilization in orotate phosphoribosyltransferases by binding isotope effects

Orotate phosphoribosyltransferases (OPRTs) form and break the N-ribosidic bond to pyrimidines by way of ribocation-like transition states (TSs) and therefore exhibit large α-secondary 1'-(3)H k(cat)/K(m) kinetic isotope effects (KIEs) [Zhang, Y., and Schramm, V. L. (2010) J. Am. Chem. Soc. 132, 8787-8794]. Substrate binding isotope effects (BIEs) with OPRTs report on the degree of ground-state destabilization for these complexes and permit resolution of binding and transition-state effects from the k(cat)/K(m) KIEs. The BIEs for interactions of [1'-(3)H]orotidine 5'-monophosphate (OMP) with the catalytic sites of Plasmodium falciparum and human OPRTs are 1.104 and 1.108, respectively. These large BIEs establish altered sp(3) bond hybridization of C1' toward the sp(2) geometry of the transition states upon OMP binding. Thus, the complexes of these OPRTs distort OMP part of the way toward the transition state. As the [1'-(3)H]OMP k(cat)/K(m) KIEs are approximately 1.20, half of the intrinsic k(cat)/K(m) KIEs originate from BIEs. Orotidine, a slow substrate for these enzymes, binds to the catalytic site with no significant [1'-(3)H]orotidine BIEs. Thus, OPRTs are unable to initiate ground-state destabilization of orotidine by altered C1' hybridization because of the missing 5'-phosphate. However the k(cat)/K(m) KIEs for [1'-(3)H]orotidine are also approximately 1.20. The C1' distortion for OMP happens in two steps, half upon binding and half on going from the Michaelis complex to the TS. With orotidine as the substrate, there is no ground-state destabilization in the Michaelis complexes, but the C1' distortion at the TS is equal to that of OMP. The large single barrier for TS formation with orotidine slows the rate of barrier crossing.     

The effective molarity of the substrate phosphoryl group in the transition state for yeast OMP decarboxylase

The second order rate constant (k(cat)/K(m)) for decarboxylation of orotidine by yeast OMP decarboxylase (ODCase), measured by trapping (14)CO(2) released during the reaction, is 2 x 10(-4)M(-1)s(-1). This very low activity may be compared with a value of 3 x 10(7)M(-1)s(-1) for the action of yeast OMP decarboxylase on the normal substrate OMP. Both activities are strongly inhibited by 6-hydroxy UMP (BMP), and abrogated by mutation of Asp-96 to alanine. These results, in conjunction with the binding affinity of inorganic phosphate as a competitive inhibitor (K(i)=7 x 10(-4)M), imply an effective concentration of 1.1 x 10(9)M for the substrate phosphoryl group in stabilizing the transition state for enzymatic decarboxylation of OMP. The observed difference in rate (1.5 x 10(11)-fold) is the largest effect of a simple substituent that appears to have been reported for an enzyme reaction.     

Role of enzyme-ribofuranosyl contacts in the ground state and transition state for orotidine 5'-phosphate decarboxylase: a role for substrate destabilization?

The crystal structure of yeast orotidine 5'-monophosphate decarboxylase (ODCase) complexed with the inhibitor 6-hydroxyuridine 5'-phosphate (BMP) reveals the presence of a series of strong interactions between enzyme residues and functional groups of this ligand. Enzyme contacts with the phosphoribofuranosyl moiety of orotidine 5'-phosphate (OMP) have been shown to contribute at least 16.6 kcal/mol of intrinsic binding free energy to the stabilization of the transition state for the reaction catalyzed by yeast ODCase. In addition to these enzyme-ligand contacts, active site residues contributed by both subunits of the dimeric enzyme are positioned to form hydrogen bonds with the 2'- and 3'-OH groups of the ligand's ribosyl moiety. These involve Thr-100 of one subunit and Asp-37 of the opposite subunit, respectively. To evaluate the contributions of these ribofuranosyl contacts to ground state and transition state stabilization, Thr-100 and Asp-37 were each mutated to alanine. Elimination of the enzyme's capacity to contact individual ribosyl OH groups reduced the k(cat)/K(m) value of the T100A enzyme by 60-fold and that of the D37A enzyme by 300-fold. Removal of the 2'-OH group from the substrate OMP decreased the binding affinity by less than a factor of 10, but decreased k(cat) by more that 2 orders of magnitude. Upon removal of the complementary hydroxymethyl group from the enzyme, little further reduction in k(cat)/K(m) for 2'-deoxyOMP was observed. To assess the contribution made by contacts involving both ribosyl hydroxyl groups at once, the ability of the D37A mutant enzyme to decarboxylate 2'-deoxyOMP was measured. The value of k(cat)/K(m) for this enzyme-substrate pair was 170 M(-1) s(-1), representing a decrease of more than 7.6 kcal/mol of binding free energy in the transition state. To the extent that electrostatic repulsion in the ground state can be tested by these simple alterations, the results do not lend obvious support to the view that electrostatic destabilization in the ground state enzyme-substrate complex plays a major role in catalysis.     

Orotidine 5'-monophosphate decarboxylase: transition state stabilization from remote protein-phosphodianion interactions

Mutants of orotidine 5'-monophosphate decarboxylase containing all possible single (Q215A, Y217F, and R235A), double, and triple substitutions of the side chains that interact with the phosphodianion group of the substrate orotidine 5'-monophosphate have been prepared. Essentially the entire effect of these mutations on the decarboxylation of the truncated neutral substrate 1-(β-d-erythrofuranosyl)orotic acid that lacks a phosphodianion group is expressed as a decrease in the third-order rate constant for activation by phosphite dianion. The results are consistent with a model in which phosphodianion binding interactions are utilized to stabilize a rare closed enzyme form that exhibits a high catalytic activity for decarboxylation.     

Uridine-5'-phosphate synthase: evidence for substrate cycling involving this bifunctional protein

Uridine 5'-phosphate (UMP) synthase contains two sequential catalytic activities for the synthesis of orotidine 5'-phosphate (OMP) from orotate (EC 2.4.2.10, orotate phosphoribosyltransferase) and the decarboxylation of OMP to form UMP (EC 4.1.1.23, OMP decarboxylase). Previous kinetic studies had indicated that partial channeling of OMP might occur [T.W. Traut and M.E. Jones (1977) J. Biol. Chem. 252, 8374-8381]; in the presence of a nucleotidase, there was no measurable formation of orotidine from OMP under conditions where OMP was maintained at a steady-state concentration [T.W. Traut (1980) Arch. Biochem. Biophys. 200, 590-594]. Recently claims were made that (i) the steady-state activities of UMP synthase could be modeled by Michaelis-Menten kinetics, and (ii) the nucleotidase activity in Ehrlich ascites cells was insufficient to degrade any significant amount of OMP [R.W. McClard and K.M. Shokat (1987) Biochemistry 26, 3378-3384]. The present studies show that UMP synthase has cooperative kinetics toward OMP, and that a substrate cycle involving orotate phosphoribosyltransferase, cytoplasmic nucleotidase, and uridine phosphorylase maintains the cyclic interconversion: orotate----OMP----orotidine----orotate, etc. It is therefore the complex steady-state kinetics of UMP synthase in the presence of OMP, and the existence of a substrate cycle that account for the results which were interpreted as channeling in the earlier studies.     

A method for assaying orotate phosphoribosyltransferase and measuring phosphoribosylpyrophosphate

An orotate phosphoribosyltransferase, OPRTase, assay method which relies upon binding reactant [3H]orotic acid and product [3H]orotidine-5'-monophosphate to polyethyleneimine-impregnated-cellulose resin and collecting on a GFC glass fiber filter is presented. Elution with 2 X 5 ml of 0.1 M sodium chloride in 5 mM ammonium acetate removes all of the orotate and leaves all of the product orotidine monophosphate (OMP) bound so that it may be measured in a scintillation counter. It was found that the addition of 10 microM barbituric acid riboside monophosphate to the reaction mixture prevented the conversion of OMP to UMP and products of UMP. The assay is suitable for measurement of OPRTase activity with purified enzyme or in crude homogenates. A modification of this scheme using commercially available yeast OPRTase and 10 microM of unlabeled OMP provides an assay for phosphoribosylpyrophosphate with a sensitivity such that 10 pmol of PRPP may be measured.     

A Raman-active competitive inhibitor of OMP decarboxylase

6-Cyanouridine 5'-phosphate was shown to act as a competitive inhibitor of yeast OMP decarboxylase, with a K(i) value of 1.1 x 10(-5)M. Upon binding by the active site of yeast OMP decarboxylase (EC 4.1.1.23), the Raman stretching frequency of the nitrile group of 6-cyanouridine 5'-phosphate decreases from 2240 to 2225 cm(-1). Based on the behavior of a model compound, 6-cyano-1,3-dimethyluracil, and on vibrational calculations, the observed change in stretching frequency is attributed to desolvation of the ligand, and distortion of the ligand in which the nitrile group moves out of the plane of the pyrimidine ring. Similar distortions may play a role in substrate activation by OMP decarboxylase, contributing to the catalytic process.     

Targeted disruption of the Myxococcus xanthus orotidine 5''-monophosphate decarboxylase gene: effects on growth and fruiting-body development.

The Myxococcus xanthus gene coding for orotidine 5'-monophosphate (OMP) decarboxylase (EC 4.1.1.23) was cloned. The M. xanthus uraA gene efficiently complemented an Escherichia coli OMP decarboxylase mutant, permitting it to grow in the absence of uracil. Electroporation of M. xanthus with a circular plasmid carrying a selectable uraA::kan gene disruption resulted in homologous recombination at the chromosomal uraA locus. Chromosomal integration of the gene disruption plasmid created heterozygous (uraA+/uraA::kan) tandem duplications. These tandem duplications were unstable and segregated auxotrophic uraA::kan daughters at frequencies of 2 x 10(-4) to 8 x 10(-4) per viable cell. Rare uraA::kan segregants were easily obtained by selecting for resistance to the toxic analog 5-fluoroorotic acid. Our experiments suggest that the cloned uraA gene could facilitate the use of gene duplications in the genetic analysis of M. xanthus development. The uraA mutants could utilize uracil, uridine, or uridine 5'-phosphate for growth, indicating that M. xanthus has pyrimidine salvage pathways. During multicellular development, uraA::kan gene disruption mutants sporulated to wild-type levels but formed smaller and more numerous aggregates than did their uraA+ parent, regardless of whether uracil was added to the medium. Pyrimidine deprivation of uraA mutants, under conditions that otherwise supported vegetative growth, failed to induce fruiting-body development or sporulation.     

Orotidine-5′-Phosphate Decarboxylase and Pyrophosphorylase of Bean Leaves

This report includes results demonstrating the existence of orotidine-5'-phosphate decarboxylase and orotidine-5'-phosphate pyrophosphorylase in plant leaves. The decarboxylase enzyme, purified 8 fold from leaves of etiolated pinto beans (Phaseolus vulgaris L.), had a pH optimum of 6.3. It was strongly inhibited by 6-azauridine-5'-phosphate; a concentration of 12 mum decreased the reaction rate 60%. The enzyme was not dependent upon magnesium ions or inhibited by p-chloromercuribenzoate. It was present in other parts of the bean plant and was found in young leaves of tomato (Lycopersicon esculentum Mill.) and Canada thistle (Cirsium arvense L.)The enzyme orotidine-5'-phosphate pyrophosphorylase, which catalyzes the formation of orotidine-5'-phosphate from orotic acid and 5-phosphoribosyl-1-pyrophosphate, was found in the etiolated bean leaves, and was also present in the leaves of tomato and Canada thistle. It was stimulated by manganous or magnesium ions and had a pH optimum of 7.2. The K(m) value obtained by varying the concentrations of 5-phosphoribosyl-1-pyrophosphate was 75 mum, and when orotic acid was varied the resulting K(m) was 3.5 mum.The presence of these 2 enzymes in higher plants, combined with previous results with inhibitors and labeled metabolites, indicates that the normal pathway of pyrimidine nucleotide synthesis in higher plants proceeds through orotic acid and OMP.     

Crystallization and preliminary X-ray crystallographic analysis of orotate phosphoribosyltransferase from Helicobacter pylori

Orotic acid phosphoribosyltransferase (PyrE) (EC 2.4.2.10) is a key enzyme in de novo uridine monophosphate (UMP) biosynthesis. It catalyzes the reaction between orotic acid and 5-phosphoribosyl-1-pyrophosphate (PRPP) to yield orotidine monophosphate (OMP), which is transformed to uridine monophosphate by decarboxylation. H. pylori PyrE was crystallized at 294 +/- 1 K by the hanging drop vapor-diffusion method. The crystals belong to the space group P2(1)2(1)2(1) with unit-cell dimensions a = 95.8, b = 104.9, c = 281.1 A, alpha = beta = gamma = 90 degrees. A set of diffraction data was collected to 3.29 A resolution using synchrotron X-ray radiation.     

Study on the Optical Rotatory Dispersion of Di-Peptides and its Application to the Recognition of Di-Peptides from Higher Peptides and Amino Acids

During the cource of the investigation of ribotidation of purine and pyrimidine bases by Brevibacterium ammoniagenes ATCC 6872, it was found that a large amount of uridine 5′-monophosphate (UMP) was accumulated in the culture broth when the organism was incubated in a medium containing uracil or orotic acid. The yields of UMP were 83% (4.8 mg/ml) from uracil and 100% (4.3 mg/ml) from orotic acid when each substrate was added at the concentration of 2 mg/ml.

Addition of 6-azauracil or 5-hydroxyuracil to the culture of the organism during cultivation led to the accumulation of both orotidine 5′-monophosphate (OMP) and UMP. The accumulation of OMP seemed to be due to the inhibition of OMP decarboxylase (E. C. 4.1.1.23) by the ribotide formed from each base. The OMP accumulation was enhanced by the addition of orotic acid in addition to 6-azauracil. When 6-azauracil was added to the medium before inoculation, UMP was predominantly accumulated, and when it was added after one day incubation, OMP was predominantly accumulated. A largest accumulation (3.6 mg/ml) of OMP was obtained when 6-azauracil was added on the 1st day and orotic acid was added on the 3rd day.

UMP and OMP accumulated in the medium were isolated from the cultured broth and identified by usual methods.     

Probing the determinants of coenzyme specificity in ferredoxin-NADP+ reductase by site-directed mutagenesis

On the basis of sequence and three-dimensional structure comparison between Anabaena PCC7119 ferredoxin-NADP(+) reductase (FNR) and other reductases from its structurally related family that bind either NADP(+)/H or NAD(+)/H, a set of amino acid residues that might determine the FNR coenzyme specificity can be assigned. These residues include Thr-155, Ser-223, Arg-224, Arg-233 and Tyr-235. Systematic replacement of these amino acids was done to identify which of them are the main determinants of coenzyme specificity. Our data indicate that all of the residues interacting with the 2'-phosphate of NADP(+)/H in Anabaena FNR are not involved to the same extent in determining coenzyme specificity and affinity. Thus, it is found that Ser-223 and Tyr-235 are important for determining NADP(+)/H specificity and orientation with respect to the protein, whereas Arg-224 and Arg-233 provide only secondary interactions in Anabaena FNR. The analysis of the T155G FNR form also indicates that the determinants of coenzyme specificity are not only situated in the 2'-phosphate NADP(+)/H interacting region but that other regions of the protein must be involved. These regions, although not interacting directly with the coenzyme, must produce specific structural arrangements of the backbone chain that determine coenzyme specificity. The loop formed by residues 261-268 in Anabaena FNR must be one of these regions.     

The in silico screening and X-ray structure analysis of the inhibitor complex of Plasmodium falciparum orotidine 5'-monophosphate decarboxylase

Orotidine 5'-monophosphate decarboxylase from Plasmodium falciparum (PfOMPDC) catalyses the final step in the de novo synthesis of uridine 5'-monophosphate (UMP) from orotidine 5'-monophosphate (OMP). A defective PfOMPDC enzyme is lethal to the parasite. Novel in silico screening methods were performed to select 14 inhibitors against PfOMPDC, with a high hit rate of 9%. X-ray structure analysis of PfOMPDC in complex with one of the inhibitors, 4-(2-hydroxy-4-methoxyphenyl)-4-oxobutanoic acid, was carried out to at 2.1 ? resolution. The crystal structure revealed that the inhibitor molecule occupied a part of the active site that overlaps with the phosphate-binding region in the OMP- or UMP-bound complexes. Space occupied by the pyrimidine and ribose rings of OMP or UMP was not occupied by this inhibitor. The carboxyl group of the inhibitor caused a dramatic movement of the L1 and L2 loops that play a role in the recognition of the substrate and product molecules. Combining part of the inhibitor molecule with moieties of the pyrimidine and ribose rings of OMP and UMP represents a suitable avenue for further development of anti-malarial drugs.     

The mechanism of orotidine 5'-monophosphate decarboxylase: catalysis by destabilization of the substrate

The mechanism of orotidine 5'-monophosphate decarboxylase (OMP decarboxylase, ODCase) was studied using the decarboxylation of orotic acid analogues as a model system. The rate of decarboxylation of 1,3-dimethylorotic acid and its analogues as well as the stability of their corresponding carbanion intermediates was determined. The results have shown that the stability of the carbanion intermediate is not a critical factor in the rate of decarboxylation. On the other hand, the reaction rate is largely dependent on the equilibrium constant for the formation of a zwitterion. Based on these results, we have proposed a new mechanism in which ODCase catalyzes the decarboxylation of OMP by binding the substrate in a zwitterionic form and providing a destabilizing environment for the carboxylate group of OMP.     

Does the bifunctional uridylate synthase channel orotidine 5'-phosphate? Kinetics of orotate phosphoribosyltransferase and orotidylate decarboxylase activities fit a noninteracting sites model

Uridylate synthase is a bifunctional protein that first forms orotidine 5'-phosphate (OMP) from orotate via its orotate phosphoribosyltransferase activity (EC 2.4.2.10) and then converts OMP to uridine 5'-phosphate (UMP) via the OMP decarboxylase activity (EC 4.1.1.23). A computer modeling analysis of the experiments that led to the proposal [Traut, T.W., & Jones, M.E. (1977) J. Biol. Chem. 252, 8374-8381] that uridylate synthase channels intermediate OMP suggests that the experimental results do not demonstrate preferential use of OMP generated in the bifunctional complex as against exogenous OMP. This analysis shows that the experimentally observed amounts of [6-14C]UMP from [6-14C]orotate in the presence of various amounts of exogenous [7-14C]OMP agree well with the amounts predicted by the computer simulations. Thus we conclude that uridylate synthase does not channel OMP. Additionally, the subsequent suggestion that channeling of OMP occurs to protect the intermediate from degradation by a nucleotidase [Traut, T.W. (1980) Arch. Biochem. Biophys. 200, 590-594] seems unlikely. The appropriate computer simulation demonstrates that low transient levels of OMP and protection of the intermediate are provided for strictly by the kinetic parameters of orotate phosphoribosyltransferase, OMP decarboxylase, and the nucleotidase. Additionally, calculations show that, in both sets of published experiments, the concentration of transient OMP greatly exceeded the concentration of OMP decarboxylase active sites. Thus, channeling of OMP by the bifunctional complex cannot be invoked to explain the evolution of uridylate synthase, and that event must be the result of some other selective pressure.     

Human lysosomal cathepsin G and granzyme B share a functionally conserved broad spectrum antibacterial peptide

Human neutrophil lysosomal cathepsin G (cat G) exerts broad-spectrum antibacterial action in vitro against Gram-negative and -positive bacteria independent of its serine protease activity. We recently determined that an internal peptide of cat G (HPQYNQR), obtained after digestion of cat G with clostripain, possessed broad-spectrum antibacterial action in vitro, displaying an ED50 of 5 x 10(-5) M. In order to evaluate the structure-antibacterial properties of this peptide, synthetic variants with single alanine substitutions at each position were prepared and tested for antibacterial action. We found that alanine substitution for His-1 or Tyr-4, or certain modifications of the His-1 side chain, produced nonbactericidal peptides. A hexapeptide lacking the COOH-terminal Arg-7 but not a pentapeptide lacking both Gln-6 and Arg-7 possessed in vitro bactericidal activity. Interestingly, the cat G bactericidal peptide displays similarity to sequences within other serine proteases, notably the proposed cytotoxic granzymes present in the cytolytic granules of human and mouse cytotoxic T lymphocytes. We now report that an internal peptide of one human granzyme (granzyme B) with the sequence of HPAYNPK also displays bactericidal action in vitro. Our results suggest that an internal antibacterial domain among human serine proteases cat G and granzyme B has been functionally conserved through evolution perhaps for the purpose of host defense against microbial pathogens and targets of cytotoxic T lymphocyte killing.     

Half-of-sites binding of orotidine 5'-phosphate and alpha-D-5-phosphorylribose 1-diphosphate to orotate phosphoribosyltransferase from Saccharomyces cerevisiae supports a novel variant of the Theorell-Chance mechanism with alternating site catalysis

A ping-pong bi-bi kinetic mechanism ascribed to yeast orotate phosphoribosyltransferase (OPRTase) [Victor, J., Greenberg, L. B., and Sloan, D. L. (1979) J. Biol. Chem. 254, 2647-2655] has been shown to be inoperative [Witte, J. F., Tsou, R., and McClard, R. W. (1999) Arch. Biochem. Biophys. 361, 106-112]. Radiolabeled orotidine 5'-phosphate (OMP), generated in situ from [7-(14)C]-orotate and alpha-d-5-phoshorylribose 1-diphosphate (PRPP), binds tightly enough to OPRTase (a dimer composed of identical subunits) that the complex survives gel-filtration chromatography. When a sample of OMP.OPRTase is extensively dialyzed, a 1:1 (per OPRTase dimer) complex is detected by (31)P NMR. Titration of the apoenzyme with OMP yields a (31)P NMR spectrum with peaks for both free and enzyme-bound OMP when OMP is in excess; the complex maintains an OMP/enzyme ratio of 1:1 even when OMP is in substantial excess. A red shift in the UV spectrum of the OMP.OPRTase complex was exploited to measure K(d(OMP)) = 0.84 muM and to verify the 1:1 binding stoichiometry. PRPP forms a Mg(2+)-dependent 1:1 complex with the enzyme as observed by (31)P NMR. Isothermal titration calorimetry (ITC) experiments revealed 1:1 stoichiometries for both OMP and Mg(2+)-PRPP with OPRTase yielding K(d) values of 0.68 and 10 microM, respectively. The binding of either 1 equiv of OMP or PRPP is mutually exclusive. ITC experiments demonstrate that the binding of OMP is largely driven by increased entropy, suggesting substantial distal disordering of the protein. Analytical gel-filtration chromatography confirms that the OMP.OPRTase complex involves the dimeric form of enzyme. The off rate for release of OMP, determined by magnetization inversion transfer, was determined to be 27 s(-)(1). This off rate is somewhat less than the k(cat) in the biosynthetic direction (about 39 s(-)(1)); thus, the release of OMP from OMP.OPRTase may not be kinetically relevant to the steady-state reaction cycle. The body of available data can be explained in terms of alternating site catalysis with either a classical Theorell-Chance mechanism or, far more likely, a novel "double Theorell-Chance" mechanism unique to alternating site catalysis, leading us to propose co-temporal binding of orotate and the release of diphosphate as well as the binding of PRPP and the release of OMP that occur via ternary complexes in alternating site fashion across the two highly cooperative subunits of the enzyme. This novel "double Theorell-Chance" mechanism yields a steady-state rate equation indistinguishable in form from the observed classical ping-pong bi-bi kinetics.     

Engineered recombinant factor VII Q217 variants with altered inhibitor specificities.

Recombinant factor VII with residue 217 (chymotrypsinogen numbering system) converted to alanine (VIIQ217A), glutamic acid (VIIQ217E), or glycine (VIIQ217G) was characterized. In a prothrombin time assay, VIIQ217E demonstrated 100%, VIIQ217A 15%, and VIIQ217G <1% clotting activities relative to wild-type VII. Binding of VIIQ217A and VIIQ217G to TF was comparable to that of wild-type VII to TF. All the variants were readily activated by factor Xa. Autoactivation in the presence of TF was efficient with VIIQ217E, slow with VIIQ217A, but undetected with VIIQ217G. Relative to wild-type VII added at the same concentration, VIIQ217E had no effect on the PT of normal plasma, whereas VIIQ217A slightly and VIIQ217G dramatically prolonged the clotting time in a dose-dependent manner. Activation of macromolecular substrates paralleled this functional inhibition. The k(cat)/K(M) values for factor X activation in the presence of TF were 2.4 for VIIaQ217E as compared to 1.9 (M(-)(1) s(-)(1) x 10(7)) for wild-type VIIa, 1.57 for VIIaQ217A, and 0.05 with VIIaQ217G. In comparison to wild-type VIIa, VIIaQ217E cleaved the chromogenic substrate S2765 (Z-D-Arg-Gly-Arg-pNA) with 10-fold higher k(cat). Analysis of the interactions with the inhibitors TFPI and antithrombin III demonstrated that VIIaQ217A but not VIIaQ217E or VIIaQ217G was inhibited less efficiently by TFPI either in the presence or in the absence of factor Xa. In contrast, VIIaQ217A association with antithrombin III in the presence of heparin was the fastest among the variants with a second-order rate constant of 2.31 (x10(3) M(-)(1) min(-)(1)), as compared to 0.47 and 1.47 for VIIaQ217E and wild-type VIIa, respectively. Our results demonstrate that residue Q(217) is important in regulating substrate and, more importantly, inhibitor recognition by VIIa.     

Hypersensitive substrate for ribonucleases.

A substrate for a hypersensitive assay of ribonucleolytic activity was developed in a systematic manner. This substrate is based on the fluorescence quenching of fluorescein held in proximity to rhodamine by a single ribonucleotide embedded within a series of deoxynucleotides. When the substrate is cleaved, the fluorescence of fluorescein is manifested. The optimal substrate is a tetranucleotide with a 5',6-carboxyfluorescein label (6-FAM) and a 3',6-carboxy-tetramethylrhodamine (6-TAMRA) label: 6-FAM-dArUdAdA-6-TAMRA. The fluorescence of this substrate increases 180-fold upon cleavage. Bovine pancreatic ribonuclease A (RNase A) cleaves this substrate with a k (cat)/ K (m)of 3.6 x 10(7)M(-1)s(-1). Human angiogenin, which is a homolog of RNase A that promotes neovascularization, cleaves this substrate with a k (cat)/ K (m)of 3. 3 x 10(2)M(-1)s(-1). This value is >10-fold larger than that for other known substrates of angio-genin. With these attributes, 6-FAM-dArUdAdA-6-TAMRA is the most sensitive known substrate for detecting ribo-nucleolytic activity. This high sensitivity enables a simple protocol for the rapid determination of the inhibition constant ( K (i)) for competitive inhibitors such as uridine 3'-phosphate and adenosine 5'-diphos-phate.     

A structure-based site-directed mutagenesis study on the neurolysin (EC 3.4.24.16) and thimet oligopeptidase (EC 3.4.24.15) catalysis

Neurolysin (EP24.16) and thimet oligopeptidase (EP24.15) are closely related metalloendopeptidases. Site-directed mutagenesis of Tyr(613) (EP24.16) or Tyr(612) (EP24.15) to either Phe or Ala promoted a strong reduction of k(cat)/K(M) for both enzymes. These data suggest the importance of both hydroxyl group and aromatic ring at this specific position during substrate hydrolysis by these peptidases. Furthermore, the EP24.15 A607G mutant showed a k(cat)/K(M) of 2x10(5) M(-1) s(-1) for the Abz-GFSIFRQ-EDDnp substrate, similar to that of EP24.16 (k(cat)/K(M)=3x10(5) M(-1) s(-1)) which contains Gly at the corresponding position; the wild type EP24.15 has a k(cat)/K(M) of 2.5x10(4) M(-1) s(-1) for this substrate.