Development of intramolecularly quenched fluorescent peptides as substrates of angiotensin-converting enzyme 2

Angiotensin-converting enzyme 2 (ACE2 or ACEH) is a novel angiotensin-converting enzyme-related carboxypeptidase that cleaves a single amino acid from angiotensin I, des-Arg bradykinin, and many other bioactive peptides. Using des-Arg bradykinin as a template, we designed a series of intramolecularly quenched fluorogenic peptide substrates for ACE2. The general structure of the substrates was F-X-Q, in which F was the fluorescent group, Abz, Q was the quenching group (either Phe(NO(2)) or Tyr(NO(2))), and X was the intervening peptide. These substrates were selectively cleaved by recombinant human ACE2, as shown by MS and HPLC. Quenching efficiency increased as the peptide sequence was shortened from 8 to 3 aa, and also when Tyr(NO(2)) was used as a quenching group instead of Phe(NO(2)). Two of the optimized substrates, TBC5180 and TBC5182, produced a signal:noise ratio of better than 20 when hydrolyzed by ACE2. Kinetic measurements with ACE2 were as follows: TBC5180, K(m)=58 microM and k(cat)/K(m)=1.3x10(5)M(-1)s(-1); TBC5182, K(m)=23 microM and k(cat)/K(m)=3.5 x 10(4)M(-1)s(-1). Thus, based on hydrolysis rate, TBC5180 was a better substrate than TBC5182. However, TBC5180 was also hydrolyzed by ACE, whereas TBC5182 was not cleaved, suggesting that TBC5182 was a selective for ACE2. We conclude that these two peptides can be used as fluorescent substrates for high-throughput screening for selective inhibitors of ACE2 enzyme.     

Production of a recombinant hybrid hemoflavoprotein: engineering a functional NADH:cytochrome c reductase.

A gene has been constructed coding for a unique fusion protein, NADH:cytochrome c reductase, that comprises the soluble heme-containing domain of rat hepatic cytochrome b(5) as the amino-terminal portion of the protein and the soluble flavin-containing domain of rat hepatic cytochrome b(5) reductase as the carboxyl terminus. The gene has been expressed in Escherichia coli resulting in the highly efficient production of a functional hybrid hemoflavoprotein which has been purified to homogeneity by a combination of ammonium sulfate precipitation, affinity chromatography on 5'-ADP agarose, and size-exclusion chromatography. The purified protein exhibited a molecular mass of approximately 46 kDa by polyacrylamide gel electrophoresis and 40,875 Da, for the apoprotein, using mass spectrometry which also confirmed the presence of both heme and FAD prosthetic groups. The fusion protein showed immunological cross-reactivity with both anti-rat cytochrome b(5) and anti-rat cytochrome b(5) reductase antibodies indicating the conservation of antigenic determinants from both native domains. Spectroscopic analysis indicated the fusion protein contained both a b-type cytochrome and flavin chromophors with properties identical to those of the native proteins. Amino-terminal and internal amino acid sequencing confirmed the identity of peptides derived from both the heme- and flavin-binding domains with sequences identical to the deduced amino acid sequence. The isolated fusion protein retained NADH:ferricyanide reductase activity (k(cat) = 8.00 x 10(2) s(-1), K(NADH)(m) = 4 microM, K(FeCN(6))(m) = 11 microM) comparable to that of that of native NADH:cytochrome b(5) reductase and also exhibited both NADH:cytochrome c reductase activity (k(cat) = 2.17 x 10(2) s(-1), K(NADH)(m) = 2 microM, K(FeCN(6))(m) = 11 microM, K(Cyt.c)(m) = 1 microM) and NADH:methemoglobin reductase activity (k(cat) = 4.40 x 10(-1) s(-1), K(NADH)(m) = 3 microM, K(mHb)(m) = 47 microM), the latter two activities indicating efficient electron transfer from FAD to heme and retention of physiological function. This work represents the first successful bacterial expression of a soluble, catalytically competent, rat hepatic cytochrome b(5)-cytochrome b(5) reductase fusion protein that retains the functional properties characteristic of the individual heme and flavin domain.     

Purification and characterization of active recombinant rat kallikrein rK9.

The rat tissue kallikrein rK9 is most abundant in the submandibular gland and the prostate. It has been successfully expressed in the Pichia pastoris yeast expression system. A full-length cDNA coding for the mature rK9 was fused in frame with yeast alpha-factor cDNA. The fusion protein was secreted into the medium with high yield without being processed by the yeast KEX2 signal peptidase. Mature rK9 was efficiently released from the fusion protein by trypsin and was purified to homogeneity by one-step affinity chromatography using soya bean trypsin inhibitor (SBTI) as affinity ligand. The identity of the recombinant enzyme was checked by N-terminal amino acid sequencing, Western blot analysis and kinetic studies. The dual trypsin- and chymotrypsin-like enzymatic specificity of rK9 was assessed by determining specificity constants (k(cat)/K(m)) for the hydrolysis of fluorogenic substrates, the peptide sequences of which were derived from proparathyroid hormone (pro-PTH) and from semenogelin-I. Our results confirmed the presence of an extended binding site in the rK9 active site. We also identified a far more sensitive substrate of this enzyme than those previously described, Abz-VKKRSARQ-EDDnp, which was hydrolysed with a catalytic efficiency k(cat)/K(m) of 420000 M(-1)s(-1). Finally, we showed that four of the five major proteins contained in secretions of rat seminal vesicles were rapidly degraded by recombinant rK9.     

Re-engineering cytochrome P450 2B11dH for enhanced metabolism of several substrates including the anti-cancer prodrugs cyclophosphamide and ifosfamide

Based on recent directed evolution of P450 2B1, six P450 2B11 mutants at three positions were created in an N-terminal modified construct termed P450 2B11dH and characterized for enzyme catalysis using five substrates. Mutant I209A demonstrated a 3.2-fold enhanced k(cat)/K(m) for 7-ethoxy-4-trifluoromethylcourmarin O-deethylation, largely due to a dramatic decrease in K(m) (0.72 microM vs. 18 microM). I209A also demonstrated enhanced selectivity for testosterone 16beta-hydroxylation over 16alpha-hydroxylation. In contrast, V183L showed a 4-fold increased k(cat) for 7-benzyloxyresorufin debenzylation and a 4.7-fold increased k(cat)/K(m) for testosterone 16alpha-hydroxylation. V183L also displayed a 1.7-fold higher k(cat)/K(m) than P450 2B11dH with the anti-cancer prodrugs cyclophosphamide and ifosfamide, resulting from a approximately 4-fold decrease in K(m). Introduction of the V183L mutation into full-length P450 2B11 did not enhance the k(cat)/K(m). Overall, the re-engineered P450 2B11dH enzymes exhibited enhanced catalytic efficiency with several substrates including the anti-cancer prodrugs.     

Expression,purification, and characterization of human enteropeptidase catalytic subunit in Escherichia coli

Enteropeptidase (synonym:enterokinase, EC 3.4.21.9) is a heterodimeric serine protease of the intestinal brush border that activates trypsinogen by highly specific cleavage of the trypsinogen activation peptide following the sequence (Asp)(4)-Lys. The DNA sequence encoding the light chain (catalytic subunit) of human enteropeptidase (GenBank Accession No. U09860) was synthesized from 26 oligonucleotides by polymerase chain reaction and cloned into plasmid pET-32a downstream to the gene of fusion partner thioredoxin immediately after the DNA sequence encoding enteropeptidase recognition site. The fusion protein thioredoxin/human enteropeptidase light chain was expressed in Escherichia coli BL21(DE3) strain in both soluble and insoluble forms. The soluble recombinant fusion protein failed to undergo autocatalytic cleavage and activation; however, autocatalytic cleavage and activation of recombinant human enteropeptidase light chain (L-HEP) were achieved by solubilization and renaturation of the fusion protein from inclusion bodies and the active L-HEP was purified on agarose-linked soybean trypsin inhibitor. The purified L-HEP cleaved the synthetic peptide substrate Gly-Asp-Asp-Asp-Asp-Lys-beta-naphthylamide with kinetic parameters K(m)=0.16 mM and k(cat)=115 s(-1) and small ester Z-Lys-SBzl with K(m)=140 microM, k(cat)=133 s(-1). L-HEP associated with soybean trypsin inhibitor slowly and small ester Z-Lys-SBzl cleavage was inhibited with K(i)(*)=2.3 nM. L-HEP digested thioredoxin/human epidermal growth factor fusion protein five times faster than equal activity units of bovine recombinant light chain (EKMax, Invitrogen) at the same conditions.     

The catalytic mode of cysteine proteinases of papain (C1) family

The Proton Inventory (PI) method has been applied in the hydrolysis of synthetic substrates by papain, chymopapain and stem bromelain, comparing also their corresponding pH-(k(cat)/K(m)) profiles, and it was found: (a) k(cat)/K(m)=k(1), and thus K(S)=k(2)/k(1) is a dynamic equilibrium constant, (b) bowed-downward PI for k(cat)/K(m) exhibiting large inverse SIE, and (c) linear PI exhibiting large normal SIE for K(S), k(2) and k(3). A novel finding of this work is that the association of substrates onto all three studied cysteine proteinases proceeds via a stepwise pathway, in contrast to purely concerted pathways found previously for both acylation and deacylation. A hydrogen bond, which seems more likely to be developed across a pK(a)-value close to 4.00, connecting [see text] (papain/chymopapain or bromelain numbering), constitutes another novelty of this work.     

Mutational evidence of transition state stabilization by serine 88 in Escherichia coli type I signal peptidase

Type I signal peptidase (SPase I) catalyzes the hydrolytic cleavage of the N-terminal signal peptide from translocated preproteins. SPase I belongs to a novel class of Ser proteases that utilize a Ser/Lys dyad catalytic mechanism instead of the classical Ser/His/Asp triad found in most Ser proteases. Recent X-ray crystallographic studies indicate that the backbone amide nitrogen of the catalytic Ser 90 and the hydroxyl side chain of Ser 88 might participate as H-bond donors in the transition-state oxyanion hole. In this work, contribution of the side-chain Ser 88 in Escherichia coli SPase I to the stabilization of the transition state was investigated through in vivo and in vitro characterizations of Ala-, Cys-, and Thr-substituted mutants. The S88T mutant maintains near-wild-type activity with the substrate pro-OmpA nuclease A. In contrast, substitution with Cys at position 88 results in more than a 740-fold reduction in activity (k(cat)) whereas S88A retains much less activity (>2440-fold decrease). Measurements of the kinetic constants of the individual mutant enzymes indicate that these decreases in activity are attributed mainly to decreases in k(cat) while effects on K(m) are minimal. Thermal inactivation and CD spectroscopic analyses indicate no global conformational perturbations of the Ser 88 mutants relative to the wild-type E. coli SPase I enzyme. These results provide strong evidence for the stabilization by Ser 88 of the oxyanion intermediate during catalysis by E. coli SPase I.     

Metabolism of cholesterol, vitamin D3 and 20-hydroxyvitamin D3 incorporated into phospholipid vesicles by human CYP27A1

CYP27A1 is a mitochondrial cytochrome P450 which can hydroxylate vitamin D3 and cholesterol at carbons 25 and 26, respectively. The product of vitamin D3 metabolism, 25-hydroxyvitamin D3, is the precursor to the biologically active hormone, 1α,25-dihydroxyvitamin D3. CYP27A1 is attached to the inner mitochondrial membrane and substrates appear to reach the active site through the membrane phase. We have therefore examined the ability of bacterially expressed and purified CYP27A1 to metabolize substrates incorporated into phospholipid vesicles which resemble the inner mitochondrial membrane. We also examined the ability of CYP27A1 to metabolize 20-hydroxyvitamin D3 (20(OH)D3), a novel non-calcemic form of vitamin D derived from CYP11A1 action on vitamin D3 which has anti-proliferative activity on keratinocytes, leukemic and myeloid cells. CYP27A1 displayed high catalytic activity towards cholesterol with a turnover number (k(cat)) of 9.8 min(-1) and K(m) of 0.49 mol/mol phospholipid (510 μM phospholipid). The K(m) value of vitamin D3 was similar for that of cholesterol, but the k(cat) was 4.5-fold lower. 20(OH)D3 was metabolized by CYP27A1 to two major products with a k(cat)/K(m) that was 2.5-fold higher than that for vitamin D3, suggesting that 20(OH)D3 could effectively compete with vitamin D3 for catalysis. NMR and mass spectrometric analyses revealed that the two major products were 20,25-dihydroxyvitamin D3 and 20,26-dihydroxyvitamin D3, in almost equal proportions. Thus, the presence of the 20-hydroxyl group on the vitamin D3 side chain enables it to be metabolized more efficiently than vitamin D3, with carbon 26 in addition to carbon 25 becoming a major site of hydroxylation. Our study reports the highest k(cat) for the 25-hydroxylation of vitamin D3 by any human cytochrome P450 suggesting that CYP27A1 might be an important contributor to the synthesis of 25-hydroxyvitamin D3, particularly in tissues where it is highly expressed.     

Purification and characterization of VanXY(C), a D,D-dipeptidase/D,D-carboxypeptidase in vancomycin-resistant Enterococcus gallinarum BM4174.

VanXY(C), a bifunctional enzyme from VanC-phenotype Enterococcus gallinarum BM4174 that catalyses D,D-peptidase and D,D-carboxypeptidase activities, was purified as the native protein, as a maltose-binding protein fusion and with an N-terminal tag containing six histidine residues. The kinetic parameters of His(6)-VanXY(C) were measured for a variety of precursors of peptidoglycan synthesis involved in resistance: for D-Ala-D-Ala, the K(m) was 3.6 mm and k(cat), 2.5 s(-1); for UDP-MurNAc-L-Ala-D-Glu-L-Lys-DAla-D-Ala (UDP-MurNAc-pentapeptide[Ala]), K(m) was 18.8 mm and k(cat) 6.2 s(-1); for D-Ala-D-Ser, K(m) was 15.5 mm and k(cat) 0.35 s(-1). His(6)-VanXYC was inactive against the peptidoglycan precursor UDP-MurNAc-L-Ala-D-Glu-L-Lys-D-Ala-D-Ser (UDP-MurNAc-pentapeptide[Ser]). The rate of hydrolysis of the terminal D-Ala of UDP-MurNAc-pentapeptide[Ala] was inhibited 30% by 2 mm D-Ala-D-Ser or UDP-MurNAc-pentapeptide[Ser]. Therefore preferential hydrolysis of substrates terminating in D-Ala would occur during peptidoglycan synthesis in E. gallinarum BM4174, leaving precursors ending in D-Ser with a lower affinity for glycopeptides to be incorporated into peptidoglycan.Mutation of an aspartate residue (Asp59) of His-tagged VanXY(C) corresponding to Asp68 in VanX to Ser or Ala, resulted in a 50% increase and 73% decrease, respectively, of the specificity constant (k(cat)/K(m)) for D-Ala-D-Ala. This situation is in contrast to VanX in which mutation of Asp68-->Ala produced a greater than 200,000-fold decrease in the substrate specificity constant. This suggests that Asp59, unlike Asp68 in VanX, does not have a pivotal role in catalysis.     

Hydrolysis of biological peptides by human angiotensin-converting enzyme-related carboxypeptidase

Human angiotensin-converting enzyme-related carboxypeptidase (ACE2) is a zinc metalloprotease whose closest homolog is angiotensin I-converting enzyme. To begin to elucidate the physiological role of ACE2, ACE2 was purified, and its catalytic activity was characterized. ACE2 proteolytic activity has a pH optimum of 6.5 and is enhanced by monovalent anions, which is consistent with the activity of ACE. ACE2 activity is increased approximately 10-fold by Cl(-) and F(-) but is unaffected by Br(-). ACE2 was screened for hydrolytic activity against a panel of 126 biological peptides, using liquid chromatography-mass spectrometry detection. Eleven of the peptides were hydrolyzed by ACE2, and in each case, the proteolytic activity resulted in removal of the C-terminal residue only. ACE2 hydrolyzes three of the peptides with high catalytic efficiency: angiotensin II () (k(cat)/K(m) = 1.9 x 10(6) m(-1) s(-1)), apelin-13 (k(cat)/K(m) = 2.1 x 10(6) m(-1) s(-1)), and dynorphin A 1-13 (k(cat)/K(m) = 3.1 x 10(6) m(-1) s(-1)). The ACE2 catalytic efficiency is 400-fold higher with angiotensin II () as a substrate than with angiotensin I (). ACE2 also efficiently hydrolyzes des-Arg(9)-bradykinin (k(cat)/K(m) = 1.3 x 10(5) m(-1) s(-1)), but it does not hydrolyze bradykinin. An alignment of the ACE2 peptide substrates reveals a consensus sequence of: Pro-X((1-3 residues))-Pro-Hydrophobic, where hydrolysis occurs between proline and the hydrophobic amino acid.     

Evaluation of superior BACE1 cleavage sequences containing unnatural amino acids

A recombinant form of BACE1 (β-site amyloid precursor protein cleaving enzyme-1) corresponding to positions 46-454 of the extracellular domain of the original membrane enzyme was prepared. The recombinant BACE1 (rBACE1) had the kinetic parameters K(m)=5.5μM and k(cat)=1719s(-1). Using several libraries of substrates containing unnatural amino acids, the specificity of rBACE1 was evaluated. LC/MS of digests derived from the libraries clarified that a dodecapeptide containing unnatural amino acids at P(2) to [Formula: see text] was a superior cleavage sequence.     

Catalytic reaction pathway for the mitogen-activated protein kinase ERK2

The structural, functional, and regulatory properties of the mitogen-activated protein kinases (MAP kinases) have long attracted considerable attention owing to the critical role that these enzymes play in signal transduction. While several MAP kinase X-ray crystal structures currently exist, there is by comparison little mechanistic information available to correlate the structural data with the known biochemical properties of these molecules. We have employed steady-state kinetic and solvent viscosometric techniques to characterize the catalytic reaction pathway of the MAP kinase ERK2 with respect to the phosphorylation of a protein substrate, myelin basic protein (MBP), and a synthetic peptide substrate, ERKtide. A minor viscosity effect on k(cat) with respect to the phosphorylation of MBP was observed (k(cat) = 10 +/- 2 s(-1), k(cat)(eta) = 0.18 +/- 0.05), indicating that substrate processing occurs via slow phosphoryl group transfer (12 +/- 4 s(-1)) followed by the faster release of products (56 +/- 4 s(-1)). At an MBP concentration extrapolated to infinity, no significant viscosity effect on k(cat)/K(m(ATP)) was observed (k(cat)/K(m(ATP)) = 0.2 +/- 0.1 microM(-1) s(-1), k(cat)/K(m(ATP))(eta) = -0.08 +/- 0.04), consistent with rapid-equilibrium binding of the nucleotide. In contrast, at saturating ATP, a full viscosity effect on k(cat)/K(m) for MBP was apparent (k(cat)/K(m(MBP)) = 2.4 +/- 1 microM(-1) s(-1), k(cat)/K(m(MBP))(eta) = 1.0 +/- 0.1), while no viscosity effect was observed on k(cat)/K(m) for the phosphorylation of ERKtide (k(cat)/K(m(ERKtide)) = (4 +/- 2) x 10(-3) microM(-1) s(-1), k(cat)/K(m(ERKtide))(eta) = -0.02 +/- 0.02). This is consistent with the diffusion-limited binding of MBP, in contrast to the rapid-equilibrium binding of ERKtide, to form the ternary Michaelis complex. Calculated values for binding constants show that the estimated value for K(d(MBP)) (/= 1.5 mM). The dramatically higher catalytic efficiency of MBP in comparison to that of ERKtide ( approximately 600-fold difference) is largely attributable to the slow dissociation rate of MBP (/=56 s(-1)), from the ERK2 active site.     

Engineering Streptomyces clavuligerus deacetoxycephalosporin C synthase for optimal ring expansion activity toward penicillin G

The deacetoxycephalosporin C synthase (DAOCS) from Streptomyces clavuligerus was engineered with the aim of enhancing the conversion of penicillin G into phenylacetyl-7-aminodeacetoxycephalosporanic acid, a precursor of 7-aminodeacetoxycephalosporanic acid, for industrial application. A single round of random mutagenesis followed by the screening of 5,500 clones identified three mutants, G79E, V275I, and C281Y, that showed a two- to sixfold increase in the k(cat)/K(m) ratio compared to the wild-type enzyme. Site-directed mutagenesis to modify residues surrounding the substrate resulted in three mutants, N304K, I305L, and I305M, with 6- to 14-fold-increased k(cat)/K(m) values. When mutants containing all possible combinations of these six sites were generated to optimize the ring expansion activity for penicillin G, the double mutant, YS67 (V275I, I305M), showed a significant 32-fold increase in the k(cat)/K(m) ratio and a 5-fold increase in relative activity for penicillin G, while the triple mutant, YS81 (V275I, C281Y, I305M), showed an even greater 13-fold increase in relative activity toward penicillin G. Our results demonstrate that this is a robust approach to the modification of DAOCS for an optimized DAOCS-penicillin G reaction.     

Substrate conformation modulates aggrecanase (ADAMTS-4) affinity and sequence specificity. Suggestion of a common topological specificity for functionally diverse proteases

Protease-substrate interactions are governed by a variety of structural features. Although the substrate sequence specificities of numerous proteases have been established, "topological specificities," whereby proteases may be classified based on recognition of distinct three-dimensional structural motifs, have not. The aggrecanase members of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family cleave a variety of proteins but do not seem to possess distinct sequence specificities. In the present study, the topological substrate specificity of ADAMTS-4 (aggrecanase-1) was examined using triple-helical or single-stranded poly(Pro) II helical peptides. Substrate topology modulated the affinity and sequence specificity of ADAMTS-4 with K(m) values indicating a preference for triple-helical structure. In turn, non-catalytic ADAMTS-4 domains were critical for hydrolysis of triple-helical and poly(Pro) II helical substrates. Comparison of ADAMTS-4 with MMP-1 (collagenase 1), MMP-13 (collagenase 3), trypsin, and thermolysin using triple-helical peptide (THP) and single-stranded peptide (SSP) substrates demonstrated that all five proteases possessed efficient "triple-helical peptidase" activity and fell into one of two categories: (k(cat)/K(m))(SSP) > (k(cat)/K(m))(THP) (thermolysin, trypsin, and MMP-13) or (k(cat)/K(m))(THP) > or = (k(cat)/K(m))(SSP) and (K(m))(SSP) > (K(m))(THP) (MMP-1 and ADAMTS-4). Overall these results suggest that topological specificity may be a guiding principle for protease behavior and can be utilized to design specific substrates and inhibitors. The triple-helical and single-stranded poly(Pro) II helical peptides represent the first synthetic substrates successfully designed for aggrecanases.     

Examining thrombin hydrolysis of the factor XIII activation peptide segment leads to a proposal for explaining the cardioprotective effects observed with the factor XIII V34L mutation

In the blood coagulation cascade, thrombin cleaves fibrinopeptides A and B from fibrinogen revealing sites for fibrin polymerization that lead to insoluble clot formation. Factor XIII stabilizes this clot by catalyzing the formation of intermolecular cross-links in the fibrin network. Thrombin activates the Factor XIII a(2) dimer by cleaving the Factor XIII activation peptide segment at the Arg(37)-Gly(38) peptide bond. Using a high performance liquid chromatography assay, the kinetic constants K(m), k(cat), and k(cat)/K(m) were determined for thrombin hydrolysis of fibrinogen Aalpha-(7-20), Factor XIII activation peptide-(28-41), and Factor XIII activation peptide-(28-41) with a Val(34) to Leu substitution. This Val to Leu mutation has been correlated with protection from myocardial infarction. In the absence of fibrin, the Factor XIII activation peptide-(28-41) exhibits a 10-fold lower k(cat)/K(m) value than fibrinogen Aalpha-(7-20). With the Factor XIII V34L mutation, decreases in K(m) and increases in k(cat) produce a 6-fold increase in k(cat)/K(m) relative to the wild-type Factor XIII sequence. A review of the x-ray crystal structures of known substrates and inhibitors of thrombin leads to a hypothesis that the new Leu generates a peptide with more extensive interactions with the surface of thrombin. As a result, the Factor XIII V34L is proposed to be susceptible to wasteful conversion of zymogen to activated enzyme. Premature depletion may provide cardioprotective effects.     

Functional expression and characterization of dipeptidyl peptidase IV from the black-bellied hornet Vespa basalis in Sf21 insect cells

The maturation of mastoparan B, the major toxin peptide in the venom of Vespa basalis, requires enzymatic cleavage of its prosequence presumably via sequential liberation of dipeptides. The putative processing enzyme, dipeptidyl peptidase IV, was expressed as a glycosylated His-tag fusion protein (rDPP-IV) via the baculovirus expression system. rDPP-IV purified by one-step nickel-affinity chromatography was verified by Western blot and LC-MS/MS analysis. The k(cat)/K(m) of rDPP-IV was determined to be in the range of 10-500 mM(-1)·S(-1) for five synthetic substrates. The optimal temperature and pH for rDPP-IV were determined to be 50 °C and pH 9. Enzymatic activity of rDPP-IV was significantly reduced by 80 and 60% in the presence of sitagliptin and phenylmethylsulfonyl fluoride respectively.     

A fluorimetric method for the determination of pepsin activity

An intramolecularly quenched fluorogenic peptide containing o-aminobenzoyl (Abz) and ethylenediamine 2,4-dinitrophenyl (Eddnp) groups at amino- and carboxyl-terminal amino acid residues, Abz-Lys-Pro-Ile-Glu-Phe-Phe-Arg-Leu-Eddnp, was hydrolyzed by purified human pepsin, gastricsin, and gastric juice uniquely at the Phe-Phe bond. Kinetic parameters determined for purified pepsin were K(m)=0.68+/-0.11 microM; k(cat)=6.3+/-0.16s(-1); k(cat)/K(m)=9.26s(-1) microM(-1); Gastricsin showed K(m)=2.69+/-0.18 microM; k(cat)=0.03+/-0.005s(-1); k(cat)/K(m)=0.011s(-1) microM(-1). Gastric juice (21 samples) from subjects without gastric disorders at endoscopy examination showed activities varying from 0.0008 to 9.72 micromolml(-1)min(-1). Pepstatin A inhibition of gastric juice enzymatic activity was complete at 3.4x10(-5)M (final concentration) inhibitor. In the proposed method the presence of a unique scissile bond in the synthetic substrate provides a direct ratio between enzymatic activity and amount of substrate hydrolyzed, and a unique step reaction facilitates the use of this assay for the determination of the activity of aspartic proteinases in biological fluids and during enzyme purification procedures.     

Inhibition of a secreted glutamic peptidase prevents growth of the fungus Talaromyces emersonii

The thermophilic filamentous fungus Talaromyces emersonii secretes a variety of hydrolytic enzymes that are of interest for processing of biomass into fuel. Many carbohydrases have been isolated and characterized from this fungus, but no studies had been performed on peptidases. In this study, two acid-acting endopeptidases were isolated and characterized from the culture filtrate of T. emersonii. One of these enzymes was identified as a member of the recently classified glutamic peptidase family and was subsequently named T. emersonii glutamic peptidase 1 (TGP1). The second enzyme was identified as an aspartyl peptidase (PEP1). TGP1 was cloned and sequenced and shown to exhibit 64 and 47% protein identity to peptidases from Aspergillus niger and Scytalidium lignocolum, respectively. Substrate profiling of 16 peptides determined that TGP1 has broad specificity with a preference for large residues in the P1 site, particularly Met, Gln, Phe, Lys, Glu, and small amino acids at P1' such as Ala, Gly, Ser, or Thr. This enzyme efficiently cleaves an internally quenched fluorescent substrate containing the zymogen activation sequence (k(cat)/K(m)=2 x 10(5) m(-1) s(-1)). Maximum hydrolysis occurs at pH 3.4 and 50 degrees C. The reaction is strongly inhibited by a transition state peptide analog, TA1 (K(i)=1.5 nM), as well as a portion of the propeptide sequence, PT1 (K(i)=32 nM). Ex vivo studies show that hyphal extension of T. emersonii in complex media is unaffected by the aspartyl peptidase inhibitor pepstatin but is inhibited by TA1 and PT1. This study provides insight into the functional role of the glutamic peptidase TGP1 for growth of T. emersonii.     

Reaction of neuronal nitric-oxide synthase with 2,6-dichloroindolphenol and cytochrome c3+: influence of the electron acceptor and binding of Ca2+-activated calmodulin on the kinetic mechanism

Binding of Ca(2+)-activated calmodulin (Ca(2+)-CaM) to neuronal nitric-oxide synthase (nNOS) increases the rate of 2,6-dichloroindolphenol (DCIP) reduction 2-3-fold and that of cytochrome c(3+) 10-20-fold. Parallel initial velocity patterns indicated that both substrates were reduced via two-half reactions in a ping-pong mechanism. Product and dead-end inhibition data with DCIP were consistent with an iso ping-pong bi-bi mechanism; however, product and dead-end inhibition studies with cytochrome c(3+) were consistent with the (two-site) ping-pong mechanism previously described for the NADPH-cytochrome P450 reductase-catalyzed reduction of cytochrome c(3+) [Sem, D., and Kasper, C. (1994) Biochemistry 33, 12012--12021]. Dead-end inhibition by 2'-adenosine monophosphate (2'AMP) was competitive versus NADPH for both electron acceptors, although the value of the slope inhibition constant, K(is), was 25-30-fold greater with DCIP as the substrate than with cytochrome c(3+). The difference in the apparent affinity of 2'AMP is proposed to result from a rapidly equilibrating isomerization step that occurs in both mechanisms prior to the binding of NADPH. Thus, initial velocity, product, and dead-end inhibition data were consistent with a di-iso ping-pong bi-bi and an iso (two-site) ping-pong mechanism for the reduction of DCIP and cytochrome c(3+), respectively. The presence Ca(2+)-CaM did not alter the proposed kinetic mechanisms. The activated cofactor had a negligible effect on (k(cat)/K(m))(NADPH), while it increased (k(cat)/K(m))(DCIP) and (k(cat)/K(m))(cytc) 4.5- and 23-fold, respectively.     

Discrimination of esterase and peptidase activities of acylaminoacyl peptidase from hyperthermophilic Aeropyrum pernix K1 by a single mutation

It has been shown that highly conserved residues that form crucial structural elements of the catalytic apparatus may be used to account for the evolutionary history of enzymes. Using saturation mutagenesis, we investigated the role of a conserved residue (Arg(526)) at the active site of acylaminoacyl peptidase from hyperthermophilic Aeropyrum pernix K1 in substrate discrimination and catalytic mechanism. This enzyme has both peptidase and esterase activities. The esterase activity of the wild-type enzyme with p-nitrophenyl caprylate as substrate is approximately 7 times higher than the peptidase activity with Ac-Leu-p-nitroanilide as substrate. However, with the same substrates, this difference was increased to approximately 150-fold for mutant R526V. A more dramatic effect occurred with mutant R526E, which essentially completely abolished the peptidase activity but decreased the esterase activity only by a factor of 2, leading to a 785-fold difference in the enzyme activities. These results provide rare examples that illustrate how enzymes can be evolved to discriminate their substrates by a single mutation. The possible structural and energetic effects of the mutations on k(cat) and K(m) of the enzyme were discussed based on molecular dynamics simulation studies.