The binding of recombinant nematode anticoagulant protein c2 (NAPc2) to either factor X or Xa is a requisite step in the pathway for the potent inhibition of VIIa tissue factor. We have used NAPc2 as a tight binding probe of human Xa to investigate protein substrate recognition by the human prothrombinase complex. NAPc2 binds with high affinity (K d ϳ1 nM) to both X and Xa in a way that does not require or occlude the active site of the enzyme. In contrast, NAPc2 is a tight binding, competitive inhibitor of protein substrate cleavage by human Xa incorporated into prothrombinase with saturating concentrations of membranes and Va. By fluorescence binding studies we show that NAPc2 does not interfere with the assembly of human prothrombinase. These are properties expected of an inhibitor that blocks protein substrate recognition by targeting extended macromolecular recognition sites (exosites) on the enzyme complex. A weaker interaction (K d ‫؍‬ 260-500 nM) observed between NAPc2 and bovine X was restored to a high affinity one in a recombinant chimeric bovine X derivative containing 25 residues from the COOH terminus of the proteinase domain of human X. This region implicated in binding NAPc2 is spatially adjacent to a site previously identified as a potential exosite. Despite the weaker interaction with bovine Xa, NAPc2 was a tight binding competitive inhibitor of protein substrate cleavage by bovine prothrombinase as well. Extended enzymic surfaces elucidated with exosite-directed probes, such as NAPc2, may define a unique region of factor Xa that is modulated following its assembly into prothrombinase and in turn determines the binding specificity of the enzyme complex for its protein substrate.The proteolytic activation of prothrombin is catalyzed by the prothrombinase complex of coagulation (2-5). Prothrombinase assembles through membrane-dependent interactions between the serine proteinase, factor Xa, and the protein cofactor, factor Va (2, 3). Although solution-phase Xa is a competent enzyme, its incorporation into prothrombinase yields a profound increase in the rate of thrombin formation (2, 3).Prothrombin is the only known protein substrate cleaved efficiently by prothrombinase (2, 6). Such stringent selectivity is not evident in the action of factor Xa on oligopeptidyl substrates, nor is the rate of peptidyl substrate hydrolysis significantly enhanced upon assembly of factor Xa into prothrombinase (7,8). Thus, the narrow and defined specificity of prothrombinase toward its protein substrate is unlikely to be solely explained by the specific recognition of residues surrounding the scissile bond by the active site of factor Xa within the enzyme complex.Mechanistic studies of bovine prothrombinase function have borne out this suggestion (9 -11). A series of studies indicate that recognition of the biological substrate is achieved through stepwise interactions of the protein substrate at an extended macromolecular recognition site (exosite) in prothrombinase removed from the catalytic site of Xa, followed b...

The proton-translocating NADH-ubiquinone oxidoreductase (complex I) is the largest and least understood respiratory complex. The intrinsic redox components (FMN and iron-sulfur clusters) reside in the promontory part of the complex. Ubiquinone is the most possible key player in proton-pumping reactions in the membrane part. Here we report the presence of three distinct semiquinone species in complex I in situ, showing widely different spin relaxation profiles. As our first approach, the semiquinone forms were trapped during the steady state NADH-ubiquinone-1 (Q1) reactions in the tightly coupled, activated bovine heart submitochondrial particles, and were named SQNf (fast-relaxing component), SQNS (slow-relaxing), and SQNx (very slow relaxing). This indicates the presence of at least three different quinone-binding sites in complex I. In the current study, special attention was placed on the SQNf, because of its high sensitivities to DeltamicroH+ and to specific complex I inhibitors (rotenone and piericidin A) in a unique manner. Rotenone inhibits the forward electron transfer reaction more strongly than the reverse reaction, while piericidine A inhibits both reactions with a similar potency. Rotenone quenched the SQNf signal at a much lower concentration than that required to quench the slower relaxing components (SQNs and SQNx). A close correlation was shown between the line shape alteration of the g// = 2.05 signal of the cluster N2 and the quenching of the SQNf signal, using two different experimental approaches: (1) changing the DeltamicroH+ poise by the oligomycin titration which decreases proton leak across the SMP membrane; (2) inhibiting the reverse electron transfer with different concentrations of rotenone. These new experimental results further strengthen our earlier proposal that a direct spin-coupling occurs between SQNf and cluster N2. We discuss the implications of these findings in connection with the energy coupling mechanism in complex .

Ribonucleotide reductases (RNRs) are key enzymes in living cells that provide the precursors of DNA synthesis. The three characterized classes of RNRs differ by their metal cofactor and their stable organic radical. We have purified to near homogeneity the enzymatically active Mn-containing RNR of Corynebacterium ammoniagenes, previously claimed to represent a fourth RNR class. N-terminal and internal peptide sequence analyses clearly indicate that the C. ammoniagenes RNR is a class Ib enzyme. In parallel, we have cloned a 10-kilobase pair fragment from C. ammoniagenes genomic DNA, using primers specific for the known class Ib RNR. The cloned class Ib locus contains the nrdHIEF genes typical for class Ib RNR operon. The deduced amino acid sequences of the nrdE and nrdF genes matched the peptides from the active enzyme, demonstrating that C. ammoniagenes RNR is composed of R1E and R2F components typical of class Ib. We also show that the Mncontaining RNR has a specificity for the NrdH-redoxin and a response to allosteric effectors that are typical of class Ib RNRs. Electron paramagnetic resonance and atomic absorption analyses confirm the presence of Mn as a cofactor and show, for the first time, insignificant amounts of iron and cobalt found in the other classes of RNR. Our discovery that C. ammoniagenes RNR is a class Ib enzyme and possesses all the highly conserved amino acid side chains that are known to ligate two ferric ions in other class I RNRs evokes new, challenging questions about the control of the metal site specificity in RNR. The cloning of the entire NrdHIEF locus of C. ammoniagenes will facilitate further studies along these lines.Ribonucleotide reductases (RNRs) 1 catalyze the reduction of ribonucleotides providing 2Ј-deoxyribonucleotides for DNA replication and repair. Three well-characterized classes of RNRs, with limited sequence similarities, have been described. They differ in their overall protein structure and cofactor requirement but have in common an allosteric regulation and the use of an organic radical to initiate catalysis through free radical chemistry (1, 2) .Apart from the similarity in mechanism, the radical chain initiator and the accompanying metal cofactor differ between the three classes. Class I enzymes (␣ 2 ␤ 2 ) contain a stable tyrosyl radical and a dinuclear iron center. Class II enzymes (␣ or ␣ 2 ) use adenosylcobalamin as cofactor and cleave it to produce a 5Ј-deoxyadenosyl radical (3, 4). The anaerobic class III enzymes (␣ 2 ␤ 2 ) possess a stable glycyl radical and an iron-sulfur cluster (5). Moreover, the different RNRs require their specific physiological reductants thioredoxin, glutaredoxin, and formate, respectively (6 -8). At the beginning of the 1990s, only these three classes of RNR were known, and they were found to cover all major branches of the tree of life. However, additional types of RNRs may remain to be discovered, and questions about non-exhaustively characterized atypical RNRs have to be answered.During the last few years, an additional operon, i...

The steady-state kinetic mechanism of ␤-amyloid precursor protein-cleaving enzyme (BACE)-catalyzed proteolytic cleavage was evaluated using product and statine-(Stat(V)) or hydroxyethylene-containing (OM99-2) peptide inhibition data, solvent kinetic isotope effects, and proton NMR spectroscopy. The noncompetitive inhibition pattern observed for both cleavage products, together with the independence of Stat(V) inhibition on substrate concentration, suggests a uni-bi-iso kinetic mechanism. According to this mechanism, the enzyme undergoes multiple conformation changes during the catalytic cycle. If any of these steps are rate-limiting to turnover, an enzyme form preceding the rate-limiting conformational change should accumulate. An insignificant solvent kinetic isotope effect (SKIE) on k cat /K m , a large inverse solvent kinetic isotope effect on k cat , and the absence of any SKIE on the inhibition onset by Stat(V) during catalysis together indicate that the ratelimiting iso-step occurs after formation of a tetrahedral intermediate. A moderately short and strong hydrogen bond (at ␦ 13.0 ppm and of 0.6) has been observed by NMR spectroscopy in the enzyme-hydroxyethylene peptide (OM99-2) complex that presumably mimics the tetrahedral intermediate of catalysis. Collapse of this intermediate, involving multiple steps and interconversion of enzyme forms, has been suggested to impose a rate limitation, which is manifested in a significant SKIE on k cat . Multiple enzyme forms and their distribution during catalysis were evaluated by measuring the SKIE on the noncompetitive (mixed) inhibition constants for the C-terminal reaction product. Large, normal SKIE values were observed for these inhibition constants, suggesting that both kinetic and thermodynamic components contribute to the K ii and K is expressions, as has been suggested for other iso-mechanism featuring enzymes. We propose that a conformational change related to the reprotonation of aspartates during or after the bond-breaking event is the rate-limiting segment in the catalytic reaction of ␤-amyloid precursor protein-cleaving enzyme, and ligands binding to other than the ground-state forms of the enzyme might provide inhibitors of greater pharmacological relevance.Extracellular amyloid deposits in brain, a characteristic feature of Alzheimer's disease, is a result of proteolytic cleavage of membrane-bound amyloid precursor protein by two enzymes, ␤-secretase and ␥-secretase. The second cleavage activity (␥-secretase) is strongly associated with the presenilin multisubunit complexes (1), whereas ␤-secretase (BACE) 1 has been identified as a novel transmembrane aspartyl protease (2-4).Although aspartyl proteases have been studied for more than 4 decades, new aspects of catalysis and inhibition continue to emerge. A substantial number of these enzymes have been identified as useful targets for chemotherapeutic intervention in human diseases (5-8), yet there has been limited success in identifying clinically relevant inhibitors; hence, it is important to explo...

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