Evaluation of comparative cytochrome P450 2B4 model by photoaffinity labeling

A homology model of rabbit CYP 2B4 was constructed on the basis of the crystallographic structure of truncated mammalian CYP 2C5/3 and bacterial soluble CYPs. To validate the CYP 2B4 homology model photoaffinity labeling was employed. Three probes (I-III) containing a photo-labile azido-group and an amino-group on opposite ends of the molecule were designed for photoaffinity labeling of the CYP 2B4 in increasing distance from the heme iron. Spectroscopic data proved probes I (the shortest) and II (a middle sized) to be coordinated with the heme iron via their amino-groups in the enzyme active center while the probe III (the longest) was not bound in this way. This binding orientation of probes I and II is in accordance with the model predicting ion-pairing of the negatively charged side chain of CYP 2B4 Asp 105 and a positively charged nitrogen located in an appropriate position in structures of probes I and II, only. The lack of heme binding of the probe III is clear from its docking into the CYP 2B4 model since no Asp 105 ion-pairing is possible. The target of photoactivated probe II, Arg 197, in a distance of about 16.5 A from the heme iron, exactly matches the position of that amino acid residue, predicted from the CYP 2B4 homology model. Moreover, using this technique, a substrate access channel has been identified. To assess the predicted substrate-binding pocket, an interaction of a specific CYP 2B4 substrate, diamantane, was examined. In "silico" docking revealed strong binding of diamantane in an orientation allowing experimentally observed C4-hydroxylation. Our homology model of CYP 2B4 is thus consistent with experimental metabolic and photoaffinity labeling data.     

Muscle Contraction : (Outline Studies in Biology) by C.R. Bagshaw Chapman & Hall; London,New York, 1982 79 pages. £2.75

On incubation of B. subtilis RM125(arg15 leuA8 r_M^? m_M^?) with DNA from alkalophilic Bacillus, the transformants (Arg⁺Leu^? or Leu^?Arg⁺) appeared at pH 10. The transformants were able to grow even at pH 7. Alkalophilic Bacillus was resistant to bacteriophages π105D1C2·1012 grown on B. subtilis 1012(r^-m_M⁺) and π105D1C2·ISMR4 grown on B. subtilis ISMR4r_M⁺r_R⁺m_M⁺m_R⁺), but the recipient B. subtilis and the transformant(Arg⁺Leu^?) were susceptible to both the of the bacteriophages. The results indicate that the transformant is a B. subtilis derivative and that alkalophilicity of alkalophilic Bacillus was transferred to B. subtilis.     

Identification of a Substrate-binding Site in a Peroxisomal β-Oxidation Enzyme by Photoaffinity Labeling with a Novel Palmitoyl Derivative

Peroxisomes play an essential role in a number of important metabolic pathways including β-oxidation of fatty acids and their derivatives. Therefore, peroxisomes possess various β-oxidation enzymes and specialized fatty acid transport systems. However, the molecular mechanisms of these proteins, especially in terms of substrate binding, are still unknown. In this study, to identify the substrate-binding sites of these proteins, we synthesized a photoreactive palmitic acid analogue bearing a diazirine moiety as a photophore, and performed photoaffinity labeling of purified rat liver peroxisomes. As a result, an 80-kDa peroxisomal protein was specifically labeled by the photoaffinity ligand, and the labeling efficiency competitively decreased in the presence of palmitoyl-CoA. Mass spectrometric analysis identified the 80-kDa protein as peroxisomal multifunctional enzyme type 2 (MFE2), one of the peroxisomal β-oxidation enzymes. Recombinant rat MFE2 was also labeled by the photoaffinity ligand, and mass spectrometric analysis revealed that a fragment of rat MFE2 (residues Trp²⁴⁹ to Arg²⁵¹) was labeled by the ligand. MFE2 mutants bearing these residues, MFE2(W249A) and MFE2(R251A), exhibited decreased labeling efficiency. Furthermore, MFE2(W249G), which corresponds to one of the disease-causing mutations in human MFE2, also exhibited a decreased efficiency. Based on the crystal structure of rat MFE2, these residues are located on the top of a hydrophobic cavity leading to an active site of MFE2. These data suggest that MFE2 anchors its substrate around the region from Trp²⁴⁹ to Arg²⁵¹ and positions the substrate along the hydrophobic cavity in the proper direction toward the catalytic center.     

Two tyrosine residues outside the editing active site in Giardia lamblia leucyl-tRNA synthetase are essential for the post-transfer editing

Leucyl-tRNA synthetase (LeuRS) is responsible for the Leu-tRNA^Leu synthesis. The connective peptide 1 (CP1) domain inserted into the Rossmann nucleotide binding fold possesses editing active site to hydrolyze the mischarged tRNA^Leu with noncognate amino acid, then to ensure high fidelity of protein synthesis. A few co-crystal structures of LeuRS with tRNA^Leu in different conformations revealed that tRNA^Leu 3′ end shuttled between synthetic and editing active sites dynamically with direct and specific interaction with the CP1 domain. Here, we reported that Y515 and Y520 outside the editing active site of CP1 domain of Giardia lamblia LeuRS (GlLeuRS) are crucial for post-transfer editing by influencing the binding affinity with mischarged tRNA^Leu. Mutations on Y515 and Y520 also decreased tRNA^Leu charging activity to various extents but had no effect on leucine activation. Our results gave some biochemical knowledge about interaction of tRNA^Leu 3′ end with the CP1 domain in archaeal/eukaryotic LeuRS.     

Molecular dynamics study of interaction and substrate channeling between neuron‐specific enolase and B‐type phosphoglycerate mutase

Phosphoglycerate mutase (PGM) and enolase are consecutive enzymes in the glycolytic pathway. We used molecular dynamics simulation to examine the interaction of human B‐type PGM (dPGM‐B) and neuron‐specific enolase (NSE). Specifically, we studied the interactions of 31 orientations of these enzymes by means of the effective energy function implicit solvation method. Interactions between active regions of the enzymes occurred preferentially, although the strongest interactions appeared to be between the back side of NSE and the active regions of dPGM‐B. Cleavage of 2PG from dPGM‐B was investigated, and the Ser¹⁴–Leu³⁰ loop of dPGM‐B is suggested as a cleavage site and, likely, another entrance site of a ligand. Substrate channeling between the enzymes was observed when NSE with its active regions Leu¹¹–Asn¹⁶, Arg⁴⁹–Lys⁵⁹, and Gly¹⁵⁵–Ala¹⁵⁸ covered the Ser¹⁴–Leu³⁰ loop of dPGM‐B. Analyses of the results make us believe that the channeling between PGM and enolase “benefits” from weak interaction. The probability of formation of channeling favorable complex is estimated to be up to 5%, while functional interaction between NSE and dPGM‐B might be as high as 20%. NSE and dPGM‐B functional interaction seems not to be isotype specific. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

In Vitro Amyloidogenic Peptides of Galectin-7: POSSIBLE MECHANISM OF AMYLOIDOGENESIS OF PRIMARY LOCALIZED CUTANEOUS AMYLOIDOSIS

Pathogenesis of primary localized cutaneous amyloidosis (PLCA) is unclear, but pathogenic relationship to keratinocyte apoptosis has been implicated. We have previously identified galectin-7, actin, and cytokeratins as the major constituents of PLCA. Determination of the amyloidogenetic potential of these proteins by thioflavin T (ThT) method demonstrated that galectin-7 molecule incubated at pH 2.0 was capable of binding to the dye, but failed to form amyloid fibrils. When a series of galectin-7 fragments containing β-strand peptides were prepared to compare their amyloidogenesis, Ser³¹-Gln⁶⁷ and Arg¹²⁰-Phe¹³⁶ were aggregated to form amyloid fibrils at pH 2.0. The rates of aggregation of Ser³¹-Gln⁶⁷ and Arg¹²⁰-Phe¹³⁶ were dose-dependent with maximal ThT levels after 3 and 48 h, respectively. Their synthetic analogs, Phe³³-Lys⁶⁵ and Leu¹²¹-Arg¹³⁴, which are both putative tryptic peptides, showed comparable amyloidogenesis. The addition of sonicated fibrous form of Ser³¹-Gln⁶⁷ or Phe³³-Lys⁶⁵ to monomeric Ser³¹-Gln⁶⁷ or Phe³³-Lys⁶⁵ solution, respectively, resulted in an increased rate of aggregation and extension of amyloid fibrils. Amyloidogenic potentials of Ser³¹-Gln⁶⁷ and Phe³³-Lys⁶⁵ were inhibited by actin and cytokeratin fragments, whereas those of Arg¹²⁰-Phe¹³⁶ and Leu¹²¹-Arg¹³⁴ were enhanced in the presence of Gly⁸⁴-Arg¹¹³, a putative tryptic peptide of galectin-7. Degraded fragments of the galectin-7 molecule produced by limited trypsin digestion, formed amyloid fibrils after incubation at pH 2.0. These results suggest that the tryptic peptides of galectin-7 released at neutral pH, may lead to amyloid fibril formation of PLCA in the intracellular acidified conditions during keratinocyte apoptosis via regulation by the galectin-7 peptide as well as actin and cytokeratins.     

Structural requirements for inhibitors of cytochromes P450 2B: assessment of the enzyme interaction with diamondoids

The series of diamondoids: adamantane, diamantane, triamantane, 2-isopropenyl-2-methyladamantane and 3-isopropenyl-3-methyldiamantane (3-IPMDIA), were employed to elucidate the molecular basis of their interaction with the active site of cytochromes P450 (CYP) of a 2B subfamily. These potent inhibitors of CYP2B enzymes were docked into the homology model of CYP2B4. Apparent dissociation constants calculated for the complexes of CYP2B4 with docked diamandoids agreed closely with the experimental data showing inhibition potency of the compounds and their binding affinity to CYP2B4. Superimposed structures of docked diamondoids mapped binding site residues. As they are mainly non-polar residues, the hydrophobicity plays the major role in the binding of diamondoids. Overlapping structure of diamondoids defined an elliptical binding cavity (5.9A inner diameter, 7.9A length) forming an angle of approximately 43 degrees with the heme plane. CYP2B specific diamondoids, namely 3-IPMDIA, showing the highest binding affinity, should be considered for a potential clinical use.     

Structural and mechanistic insights into the interaction of cytochrome P4503A4 with bromoergocryptine, a type I ligand

Cytochrome P4503A4 (CYP3A4), a major human drug-metabolizing enzyme, is responsible for the oxidation and clearance of the majority of administered drugs. One of the CYP3A4 substrates is bromoergocryptine (BEC), a dopamine receptor agonist prescribed for the inhibition of prolactin secretion and treatment of Parkinson disease, type 2 diabetes, and several other pathological conditions. Here we present a 2.15 Å crystal structure of the CYP3A4-BEC complex in which the drug, a type I heme ligand, is bound in a productive mode. The manner of BEC binding is consistent with the in vivo metabolite analysis and identifies the 8′ and 9′ carbons of the proline ring as the primary sites of oxidation. The crystal structure predicts the importance of Arg²¹² and Thr²²⁴ for binding of the tripeptide and lysergic moieties of BEC, respectively, which we confirmed experimentally. Our data support a three-step BEC binding model according to which the drug binds first at a peripheral site without perturbing the heme spectrum and then translocates into the active site cavity, where formation of a hydrogen bond between Thr²²⁴ and the N1 atom of the lysergic moiety is followed by a slower conformational readjustment of the tripeptide group modulated by Arg²¹².     

A fish bradykinin (Arg0, Trp5, Leu8-bradykinin) from the defensive skin secretion of the European edible frog, Pelophylax kl. esculentus: structural characterization; molecular cloning of skin kininogen cDNA and pharmacological effects on mammalian smooth muscle

Extensive studies on bradykinin-related peptides (BRPs) generated from plasma kininogens in representative species of various vertebrate taxa, have confirmed that many amphibian skin BRPs reflect those present in putative vertebrate predators. For example, the (Val¹, Thr⁶)-bradykinin, present in the defensive skin secretions of many ranids and phyllomedusines, can be generated from plasma kininogens in colubrid snakes—common predators of these frogs. Here, we report the presence of (Arg⁰, Trp⁵, Leu⁸)-bradykinin in the skin secretion of the European edible frog, Pelophylax kl. esculentus, and have found it to be encoded in single copy by a kininogen with an open-reading frame of 68 amino acid residues. This peptide is the archetypal bony fish bradykinin that has been generated from plasma kininogens of the bowfin (Amia calva), the long-nosed gar (Lepisosteus oseus) and the rainbow trout (Onchorhynchus mykiss). More recently, this peptide has been shown to be encoded within cloned kininogens of the Atlantic cod (Gadus morhua) spotted wolf-fish (Anarichas minor), zebrafish (Danio rerio), pufferfish (Tetraodon nigroviridis) and Northern pike (Esox lucius). The latter species is regarded as a major predator of P. kl. esculentus. Synthetic (Arg⁰, Trp⁵, Leu⁸)-bradykinin was previously reported as having multiphasic effects on arterial blood pressure in conscious trout and here we have demonstrated that it can antagonize the relaxation in rat arterial smooth muscle induced by canonical mammalian bradykinin. The discovery of (Arg⁰, Trp⁵, Leu⁸)-bradykinin in the defensive skin secretion of this amphibian completes the spectrum of vertebrate taxon-specific BRPs identified from this source.     

Structural determinants of product specificity of sucrose isomerases

The healthy sweetener isomaltulose is industrially produced from the conversion of sucrose by the sucrose isomerase SmuA from Protaminobacter rubrum. Crystal structures of SmuA in native and deoxynojirimycin complexed forms completed with modeling studies unravel the characteristics of the isomaltulose synthases catalytic pocket and their substrate binding mode. Comparison with the trehalulose synthase MutB highlights the role of Arg²⁹⁸ and Arg³⁰⁶ active site residues and surface charges in controlling product specificity of sucrose isomerases (isomaltulose versus trehalulose). The results provide a rationale for the specific design of optimized enzymes.     

CYP2E1 active site residues in substrate recognition sequence 5 identified by photoaffinity labeling and homology modeling

Despite its biological importance, our knowledge of active site structure and relevance of critical amino acids in CYP2E1 catalytic processes remain limited. In this study, we identified CYP2E1 active site residues using photoaffinity labeling with 7-azido-4-methylcoumarin (AzMC) coupled with a CYP2E1 homology model. In the absence of light, AzMC was an effective competitor against substrate p-nitrophenol oxidation by CYP2E1. Photoactivation of AzMC led to a concentration-dependent loss in CYP2E1 activity and structural integrity resulting from the modification of both heme and protein. The photo-labeling reaction degraded heme and produced a possible heme adduct. Probe incorporation into the protein occurred at multiple sites within substrate recognition sequence 5 (SRS-5). Based on a CYP2E1 homology model, we hypothesize AzMC labels SRS-5 residues, Leu363, Val364, and Leu368, in the active site. In addition, we propose a series of phenylalanines, especially Phe106, mediate contacts with the coumarin.     

Heterobifunctional photoaffinity probes for cytochrome P450 2B.

Three heterobifunctional photoaffinity probes, N-(p-azidobenzyl)-N-methyl-p-aminobenzylamine (I), N-(p-azidobenzyl)-N-methyl-p-aminophenethylamine (II), and N-(p-azidophenethyl)-N-methyl-p-aminophenethylamine (III), were synthesized and characterized. These probes, containing a photolabile azido-group and an amino-group on opposite sides of the molecule, were designed for photoaffinty labeling of the cytochrome P450 (CYP) 2B active site cavity differing in distance from the heme iron. Spectroscopic studies proved that probes I and II coordinated with the heme iron via their amino-group in the enzyme active center, whereas probe III did not. This result in conjunction with data from kinetic studies suggests probes I and II are appropriate for photoaffinity labeling of the CYP 2B active center. Thus, probe II was used to identify amino acid residues within a distance of the probe length (about 16.5 A) from the heme. Analysis of a Lys-C digest of the probe II-labeled CYP 2B4 revealed a single labeled hexapeptide corresponding to position 192-197 of the CYP 2B4 sequence. Using postsource decay/matrix-assisted laser desorption ionization-time of flight, Arg197 was identified as a probe II target. The location of the labeled site in three-dimensional structures of bacterial CYPs and in CYP 2B homology models is discussed.     

Insights into binding modes of tumstatin peptide T7 with the active site of αvβ3 integrin

Through interaction with the active site of α_vβ₃ integrin, tumstatin T7 peptide inhibits both the angiogenesis and the proliferation of tumour cells. In this work, docking in conjunction with molecular dynamics simulation was used to explore the binding mode of T7 peptide and α_vβ₃ integrin. The binding mode analysis revealed that the residues Ser⁹⁰, Arg⁹¹, Asp⁹³ and Tyr⁹⁴ in T7 peptide, and (α)-Asp¹⁵⁰, (β)-Arg²¹⁴, (α)-Asp¹⁴⁸ (α)-Gln²¹⁴ and (α)-Glu¹²³ in the active site of α_vβ₃ integrin were most likely the key interaction sites. The hydroxyl of Tyr⁹⁴ coordinates α_vβ₃ via a Mn²⁺ ion, revealing that Mn²⁺ is also an important factor for the interaction. The insight into these key interaction sites not only suggests that the active site of α_vβ₃ integrin can bind to molecules through multiple binding mechanisms, but also provides some useful information for structure-based drug design.     

Fluorescent photoaffinity labeling of cytochrome P450 3A4 by lapachenole: identification of modification sites by mass spectrometry

While photoaffinity ligands (PALs) have been widely used to probe the structures of many receptors and transporters, their effective use in the study of membrane-bound cytochrome P450s is less established. Here, lapachenole has been used as an effective photoaffinity ligand of human P450 3A4, and mass spectrometry data demonstrating the efficient and specific photoaffinity labeling of CYP3A4 by this naturally occurring benzochromene compound is presented. Without photolysis, lapachenole is a substrate of CYP3A4 and can be metabolized to hydroxylated products by this enzyme. A high-performance liquid chromatography/electrospray ionization mass spectrometry (HPLC/ESI-MS) procedure was developed to analyze small amounts of intact purified CYP3A4, and analysis of the labeled protein showed the presence of one molecule of lapachenole bound per monomer of protein. Photolabeled CYP3A4 peptide adducts were further characterized by mass spectrometric analysis after proteolytic digestion and isolation of fluorescent photolabeled peptides. Two peptide adducts accounting for >95% of the labeled peptides were isolated by HPLC, and both peptides, ECYSVFTNR (positions 97-105) and VLQNFSFKPCK (positions 459-469), were identified by nano-LC/ESI quadrupole time-of-flight (QTOF) and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. The sites of modification were further localized to positions Cys-98 and Cys-468 for each peptide by nano-LC/ESI QTOF tandem mass spectrometry (MS/MS). The results provided the first direct evidence for interaction between the PAL and the putative B-B' loop region, which may serve as a substrate access channel or as a part of the CYP3A4 active site. In conclusion, benzochromene analogues are effective PALs, which may be used in the study of other cytochrome P450 structures.     

Photoaffinity labeling of peptide binding sites of prolyl 4-hydroxylase with N-(4-azido-2-nitrophenyl)glycyl-(Pro-Pro-Gly)5

The synthesis is described of the photoaffinity label N-(4-azido-2-nitrophenyl)glycyl-(Pro-Pro-Gly)5 for the peptide binding site of prolyl 4-hydroxylase. The photoaffinity label is a good substrate and is capable of light-induced inactivation of prolyl 4-hydroxylase activity. Inactivation depends on the concentration of photoaffinity label and is prevented by competition with excess (Pro-Pro-Gly)5. Two moles of photoaffinity label per mole of enzyme is needed for 100% inactivation of enzymic activity. Oxidative decarboxylation of 2-oxoglutarate measured in the absence of added peptide substrate is not affected by labeling. We conclude that the covalently bound nitreno derivative of N-(4-azido-2-nitrophenyl)glycyl-(Pro-Pro-Gly)5 acts by preventing the binding of peptide substrate to the catalytic site without interfering with the binding of the other substrates and cofactors 2-oxoglutarate, O2, Fe2+, and ascorbate. Labeling is specific for the alpha subunit of the tetrameric alpha 2 beta 2 enzyme. In addition to two catalytic binding sites that are blocked by the photoaffinity label, the enzyme contains binding subsites for peptide substrates, as judged from the capability of photoinactivated enzyme to bind to a poly(L-proline) affinity column. These binding subsites may account for the rapidly increasing affinity for peptide substrates with increasing chain length.     

Regulated Cleavage of Prothrombin by Prothrombinase: REPOSITIONING A CLEAVAGE SITE REVEALS THE UNIQUE KINETIC BEHAVIOR OF THE ACTION OF PROTHROMBINASE ON ITS COMPOUND SUBSTRATE*?

Prothrombinase converts prothrombin to thrombin via cleavage at Arg³²⁰ followed by cleavage at Arg²⁷¹. Exosite-dependent binding of prothrombin to prothrombinase facilitates active site docking by Arg³²⁰ and initial cleavage at this site. Precise positioning of the Arg³²⁰ site for cleavage is implied by essentially normal cleavage at Arg³²⁰ in recombinant prothrombin variants bearing additional Arg side chains either one or two residues away. However, mutation of Arg³²⁰ to Gln reveals that prothrombinase can cleave prothrombin following Arg side chains shifted by as many as two residues N-terminal to the 320 position at near normal rates. Further repositioning leads to a loss in cleavage at this region with an abrupt shift toward slow cleavage at Arg²⁷¹. In contrast, the binding constant for the active site docking step is strongly dependent on the sequence preceding the scissile bond as well as position. Large effects on binding only yield minor changes in rate until the binding constant passes a threshold value. This behavior is expected for a substrate that can engage the enzyme through mutually exclusive active site docking reactions followed by cleavage to yield different products. Cleavage site specificity as well as the ordered action of prothrombinase on its compound substrate is regulated by the thermodynamics of active site engagement of the individual sites as well as competition between alternate cleavage sites for active site docking.     

Inhibition of CYP2B4 by the mechanism-based inhibitor 2-ethynylnaphthalene: inhibitory potential of 2EN is dependent on the size of the substrate

2-Ethynylnaphthalene (2EN) is a mechanism-based inhibitor of CYP2B4 with two components to the inhibition, (1) enzyme inactivation, which requires covalent binding of the 2EN metabolite, and (2) reversible inhibition by 2EN itself. Both inhibitory components were examined using several different CYP2B4 substrates. Preincubation of CYP2B4 with 2EN led to a time-dependent inactivation of each of the CYP2B4-dependent activities examined; however, the ability of 2EN to reversibly inhibit CYP2B4 depended on the substrate employed, which is inconsistent with classical inhibition patterns. The degree 2EN's reversible inhibition was shown not to correlate with the substrate affinity for the active site, but with parameters related to the molecular size of the substrate. The results are consistent with 2EN and the smaller substrates simultaneously fitting in the CYP2B4 active site, leading to very little inhibition. Larger substrates exhibited greater degrees of inhibition because of their inability to co-bind with inhibitor in the active site.     

DNA haplotype-dependent differences in the amino acid sequence of debrisoquine 4-hydroxylase (CYP2D6): evidence for two major allozymes in extensive metabolisers

The molecular basis for DNA haplotype-dependent debrisoquine 4-hydroxylase (CYP2D6) expression was explored by sequencing all of the nine exons of the CYP2D6 gene. Two distinct exon sequence frameworks of the CYP2D6 gene were found, each associated with specific BamHI-defined DNA haplotypes of the CYP2D cluster. They corresponded to Arg²⁹⁶/Cys²⁹⁶ and Ser⁴⁸⁶/Thr⁴⁸⁶ amino acid polymorphisms in the CYP2D6 enzyme, and occurred in almost equal frequency among the Caucasians examined. These two major allozymes with amino acid differences in the presumed substrate recognition region and in the vicinity of the heme binding site could be the source of the observed DNA haplotype-dependent variation in phenotypic expression.     

Probing the CYP3A4 active site by cysteine scanning mutagenesis and photoaffinity labeling

The mechanism of CYP3A4-substrate interactions has been investigated using a battery of techniques including cysteine scanning mutagenesis, photoaffinity labeling, and structural modeling. In this study, cysteine scanning mutagenesis was performed at seven sites within CYP3A4 proposed to be involved in substrate interaction and/or cooperativity. Photolabeled CYP3A4 peptide adducts were further characterized by mass spectrometric analysis for each mutant after proteolytic digestion and isolation of fluorescent photolabeled peptides. Among the tryptic peptides of seven tested mutants, three photolabeled peptides of the F108C mutant, ECYSVFTNR (positions 97-105), VLQNFSFKPCK (positions 459-469), and RPCGPVGFMK (positions 106-115) were identified by MALDI-TOF-MS and nano-LC/ESI QTOF MS. The site of modification was further localized to the substituted Cys-108 residue in the mutant peptide adduct RPCGPVGFMK (positions 106-115) by nano-LC/ESI QTOF MS/MS. In summary, we described a potentially useful method to study P450 active sites using a combination of cysteine scanning mutagenesis and photoaffinity labeling.     

Inhibition of Thrombin Formation by Active Site Mutated (S360A) Activated Protein C

Activated protein C (APC) down-regulates thrombin formation through proteolytic inactivation of factor Va (FVa) by cleavage at Arg⁵⁰⁶ and Arg³⁰⁶ and of factor VIIIa (FVIIIa) by cleavage at Arg³³⁶ and Arg⁵⁶². To study substrate recognition by APC, active site-mutated APC (APC(S360A)) was used, which lacks proteolytic activity but exhibits anticoagulant activity. Experiments in model systems and in plasma show that APC(S360A), and not its zymogen protein C(S360A), expresses anticoagulant activities by competing with activated coagulation factors X and IX for binding to FVa and FVIIIa, respectively. APC(S360A) bound to FVa with a K_D of 0.11 ± 0.05 nm and competed with active site-labeled Oregon Green activated coagulation factor X for binding to FVa. The binding of APC(S360A) to FVa was not affected by protein S but was inhibited by prothrombin. APC(S360A) binding to FVa was critically dependent upon the presence of Arg⁵⁰⁶ and not Arg³⁰⁶ and additionally required an active site accessible to substrates. Inhibition of FVIIIa activity by APC(S360A) was >100-fold less efficient than inhibition of FVa. Our results show that despite exosite interactions near the Arg⁵⁰⁶ cleavage site, binding of APC(S360A) to FVa is almost completely dependent on Arg⁵⁰⁶ interacting with APC(S360A) to form a nonproductive Michaelis complex. Because docking of APC to FVa and FVIIIa constitutes the first step in the inactivation of the cofactors, we hypothesize that the observed anticoagulant activity may be important for in vivo regulation of thrombin formation.