Selective neurotensin-derived internally quenched fluorogenic substrates for neurolysin (EC 3.4.24.16): comparison with thimet oligopeptidase (EC 3.4.24.15) and neprilysin (EC 3.4.24.11)

Internally quenched fluorescent peptides derived from neurotensin (pELYENKPRRPYIL) sequence were synthesized and assayed as substrates for neurolysin (EC 3.4.24.16), thimet oligopeptidase (EC 3.4.24.15 or TOP), and neprilysin (EC 3.4.24.11 or NEP). Abz-LYENKPRRPYILQ-EDDnp (where EDDnp is N-(2,4-dinitrophenyl)ethylenediamine and Abz is ortho-aminobenzoic acid) was derived from neurotensin by the introduction of Q-EDDnp at the C-terminal end of peptide and by the substitution of the pyroglutamic (pE) residue at N-terminus for Abz and a series of shorter peptides was obtained by deletion of amino acids residues from C-terminal, N-terminal, or both sides. Neurolysin and TOP hydrolyzed the substrates at P--Y or Y--I or R--R bonds depending on the sequence and size of the peptides, while NEP cleaved P-Y or Y-I bonds according to its S'(1) specificity. One of these substrates, Abz-NKPRRPQ-EDDnp was a specific and sensitive substrate for neurolysin (k(cat) = 7.0 s(-1), K(m) = 1.19 microM and k(cat)/K(m) = 5882 mM(-1). s(-1)), while it was completely resistant to NEP and poorly hydrolyzed by TOP and also by prolyl oligopeptidase (EC 3.4.21.26). Neurolysin concentrations as low as 1 pM were detected using this substrate under our conditions and its analogue Abz-NKPRAPQ-EDDnp was hydrolyzed by neurolysin with k(cat) = 14.03 s(-1), K(m) = 0.82 microM, and k(cat)/K(m) = 17,110 mM(-1). s(-1), being the best substrate so far described for this peptidase.     

Enzyme inhibition assays using fluorescence correlation spectroscopy: a new algorithm for the derivation of kcat/KM and Ki values at substrate concentrations much lower than the Michaelis constant

A new mathematical formalism is deduced which allows for the calculation of the k(cat) over K(M) ratio based on measurements of the enzyme kinetics with substrate concentrations much lower than K(M). The equations are also applied on the action of an inhibitor on enzyme activity yielding the binding constant, K(i), of an inhibitor molecule. For practical evaluation of the new theoretical approach, the enzymatic reaction of CD45 phosphatase was used as a well-characterized model system with known inhibitors for testing the K(i) value determination scheme. The k(cat)/K(M) ratio was calulated to be 4.7 x 10(5) M(-)(1) s(-)(1), the K(i) of the inhibitor molecule PKF52-524 was estimated to be (1-2) x 10(-)(7) M and the association rate of the inhibitor PKF52-524 to CD45 phosphatase was estimated to be 59 M(-)(1) s(-)(1).     

Cloning and characterization of histamine dehydrogenase from Nocardioides simplex

Histamine dehydrogenase (NSHADH) can be isolated from cultures of Nocardioides simplex grown with histamine as the sole nitrogen source. A previous report suggested that NSHADH might contain the quinone cofactor tryptophan tryptophyl quinone (TTQ). Here, the hdh gene encoding NSHADH is cloned from the genomic DNA of N. simplex, and the isolated enzyme is subjected to a full spectroscopic characterization. Protein sequence alignment shows NSHADH to be related to trimethylamine dehydrogenase (TMADH: EC 1.5.99.7), where the latter contains a bacterial ferredoxin-type [4Fe-4S] cluster and 6-S-cysteinyl FMN cofactor. NSHADH has no sequence similarity to any TTQ containing amine dehydrogenases. NSHADH contains 3.6+/-0.3 mol Fe and 3.7+/-0.2 mol acid labile S per subunit. A comparison of the UV/vis spectra of NSHADH and TMADH shows significant similarity. The EPR spectrum of histamine reduced NSHADH also supports the presence of the flavin and [4Fe-4S] cofactors. Importantly, we show that NSHADH has a narrow substrate specificity, oxidizing only histamine (K(m)=31+/-11 microM, k(cat)/K(m)=2.1 (+/-0.4)x10(5)M(-1)s(-1)), agmatine (K(m)=37+/-6 microM, k(cat)/K(m)=6.0 (+/-0.6)x10(4)M(-1)s(-1)), and putrescine (K(m)=1280+/-240 microM, k(cat)/K(m)=1500+/-200 M(-1)s(-1)). A kinetic characterization of the oxidative deamination of histamine by NSHADH is presented that includes the pH dependence of k(cat)/K(m) (histamine) and the measurement of a substrate deuterium isotope effect, (D)(k(cat)/K(m) (histamine))=7.0+/-1.8 at pH 8.5. k(cat) is also pH dependent and has a reduced substrate deuterium isotope of (D)(k(cat))=1.3+/-0.2.     

Kinetic mechanism of an aldehyde reductase of Saccharomyces cerevisiae that relieves toxicity of furfural and 5-hydroxymethylfurfural

An effective means of relieving the toxicity of furan aldehydes, furfural (FFA) and 5-hydroxymethylfurfural (HMF), on fermenting organisms is essential for achieving efficient fermentation of lignocellulosic biomass to ethanol and other products. Ari1p, an aldehyde reductase from Saccharomyces cerevisiae, has been shown to mitigate the toxicity of FFA and HMF by catalyzing the NADPH-dependent conversion to corresponding alcohols, furfuryl alcohol (FFOH) and 5-hydroxymethylfurfuryl alcohol (HMFOH). At pH 7.0 and 25°C, purified Ari1p catalyzes the NADPH-dependent reduction of substrates with the following values (k(cat) (s(-1)), k(cat)/K(m) (s(-1)mM(-1)), K(m) (mM)): FFA (23.3, 1.82, 12.8), HMF (4.08, 0.173, 23.6), and dl-glyceraldehyde (2.40, 0.0650, 37.0). When acting on HMF and dl-glyceraldehyde, the enzyme operates through an equilibrium ordered kinetic mechanism. In the physiological direction of the reaction, NADPH binds first and NADP(+) dissociates from the enzyme last, demonstrated by k(cat) of HMF and dl-glyceraldehyde that are independent of [NADPH] and (K(ia)(NADPH)/k(cat)) that extrapolate to zero at saturating HMF or dl-glyceraldehyde concentration. Microscopic kinetic parameters were determined for the HMF reaction (HMF+NADPH?HMFOH+NADP(+)), by applying steady-state, presteady-state, kinetic isotope effects, and dynamic modeling methods. Release of products, HMFOH and NADP(+), is 84% rate limiting to k(cat) in the forward direction. Equilibrium constants, [NADP(+)][FFOH]/[NADPH][FFA][H(+)]=5600×10(7)M(-1) and [NADP(+)][HMFOH]/[NADPH][HMF][H(+)]=4200×10(7)M(-1), favor the physiological direction mirrored by the slowness of hydride transfer in the non-physiological direction, NADP(+)-dependent oxidation of alcohols (k(cat) (s(-1)), k(cat)/K(m) (s(-1)mM(-1)), K(m) (mM)): FFOH (0.221, 0.00158, 140) and HMFOH (0.0105, 0.000104, 101).     

Constitutive expression, purification and characterization of a phosphoglucomutase from Fusarium oxysporum

The phosphoglucomutase gene from a wild type Fusarium oxysporum strain (F3), was homologously expressed, under the control of the constitutive promoter of gpdA of Aspergillus nidulans. The transformant produced elevated levels of phosphoglucomutase activity compared to the wild type, a fact that facilitated the subsequent purification procedure. The enzyme (FoPGM) was purified to homogeneity applying three anion exchange and one gel filtration chromatography steps. The native enzyme revealed a monomeric structure with a molecular mass of 60 kDa, while the isoelectric point was 3.5. FoPGM was active in pH ranged from 6.0 to 8.0, with an optimum using 3-(N-morpholino)propanesulfonic acid buffer at 7.0, while loss of activity was observed when phosphate buffer was used in the above mentioned pH range. The optimal temperature for activity was 45°C but the enzyme became unstable at temperatures above 40°C. FoPGM requires the presence of a divalent cation for its function with maximum activity being obtained with Co(2+). The apparent K(m) for Co(2+) was found to be 10 μM. The enzyme was also active with other divalent metal ions such as Mn(2+), Mg(2+), Ni(2+) and Ca(2+) but to a lesser extent. The following kinetic constants were determined: v(max), 0.74 μmol mg(protein)(-1)min(-1); k(cat), 44.2 min(-1); K(m)(G1P), 0.10mM; K(m)(G1,6 diP), 1.03 μM; k(cat)/K(m)(G1P), 443 mM(-1)min(-1) and k(cat)/K(m)(G1,6 diP), 42,860 mM(-1)min(-1). The enzyme was considered to follow a Ping Pong substituted enzyme or enzyme isomerization mechanism.     

Relationship of exo-beta-D-galactofuranosidase kinetic parameters to the number of phosphodiesters in Penicillium fellutanum peptidophosphogalactomannan: enzyme purification and kinetics of glycopeptide and galactofuran chain hydrolysis

Extracellular Penicillium fellutanum exo-beta-D-galactofuranosidase, with a mass of 70 kDa, was purified to apparent homogeneity. The enzyme was used to investigate the influence of phosphodiesters of the peptidophosphogalactomannans pP(2)GM(ii) and pP(25)GM(ii) (containing 2 and 25 phosphodiester residues, respectively, per mol of polymer) on the kinetic parameters of galactofuranosyl hydrolysis of these two polymers, of 1-O-methyl-beta-D-galactofuranoside, and of two galactofuranooligosaccharides. The enzyme did not hydrolyze phosphorylated galactose residues of pP(2)GM(ii) or pP(25)GM(ii). The k(cat)/K(m) value for pP(25)GM(ii) is 1.7 x 10(3) M(-1) s(-1), that for 1-O-methyl-beta-D-galactofuranoside is 1.1 x 10(4) M(-1) s(-1), that for pP(2)GM(ii) is 1.7 x 10 (4) M(-1) s(-1), and those for 5-O-beta-D-galactofuranooligosaccharides with degrees of polymerization of 3.4 and 5.5 are 1.7 x 10(5) and 4.1 x 10(5) M(-1) s(-1), respectively. Variability in the k(cat)/K(m) values is due primarily to differences in K(m) values; the k(-1)/k(1) ratio likely provides the most influence on K(m). k(cat) increases as the degree of polymerization of galactofuranosyl residues increases. Most of the galactofuranosyl and phosphocholine residues were removed by day 8 in vivo from pP(x)GM(ii) added to day 3 cultures initiated in medium containing 2 mM phosphate but not from those initially containing 20 mM phosphate. The filtrates from day 9 cultures initiated in 2 mM inorganic phosphate in modified Raulin-Thom medium contained 0.2 mM inorganic phosphate and 2.2 U of galactofuranosidase ml(-1)h(-1). No galactofuranosidase activity but 15 mM inorganic phosphate was found in filtrates from day 9 cultures initiated in 20 mM phosphate. In vivo the rate of galactofuranosyl hydrolysis of pP(x)GM(ii) and of related polymers is proportional to the k(cat)/K(m) value of each polymer. The kinetic data show that the k(cat)/K(m) value increases as the number of phosphodiesters of pP(x)GM(ii) decreases, also resulting in an increase in the activity of exo-beta-D-galactofuranosidase.     

Rapid phosphotransfer to CheY from a CheA protein lacking the CheY-binding domain

The histidine protein kinase CheA plays a central role in the bacterial chemotaxis signal transduction pathway. Autophosphorylated CheA passes its phosphoryl group to CheY very rapidly (k(cat) approximately 750 s(-)(1)). Phospho-CheY in turn influences the direction of flagellar rotation. The autophosphorylation site of CheA (His(48)) resides in its N-terminal P1 domain. The adjacent P2 domain provides a high-affinity binding site for CheY, which might facilitate the phosphotransfer reaction by tethering CheY in close proximity to the phosphodonor located in P1. To explore the contribution of P2 to the CheA --> CheY phosphotransfer reaction in the Escherichia coli chemotaxis system, we examined the transfer kinetics of a mutant CheA protein (CheADeltaP2) in which the 98 amino acid P2 domain had been replaced with an 11 amino acid linker. We used rapid-quench and stopped-flow fluorescence experiments to monitor phosphotransfer to CheY from phosphorylated wild-type CheA and from phosphorylated CheADeltaP2. The CheADeltaP2 reaction rates were significantly slower and the K(m) value was markedly higher than the corresponding values for wild-type CheA. These results indicate that binding of CheY to the P2 domain of CheA indeed contributes to the rapid kinetics of phosphotransfer. Although phosphotransfer was slower with CheADeltaP2 (k(cat)/K(m) approximately 1.5 x 10(6) M(-)(1) s(-)(1)) than with wild-type CheA (k(cat)/K(m) approximately 10(8) M(-)(1) s(-)(1)), it was still orders of magnitude faster than the kinetics of CheY phosphorylation by phosphoimidazole and other small molecule phosphodonors (k(cat)/K(m) approximately 5-50 M(-)(1) s(-)(1)). We conclude that the P1 domain of CheA also makes significant contributions to phosphotransfer rates in chemotactic signaling.     

The role of the conserved Lys68*:Glu265 intersubunit salt bridge in aspartate aminotransferase kinetics: multiple forced covariant amino acid substitutions in natural variants

The role of the Lys68*:Glu265 intersubunit salt bridge that is conserved (Csb) in all known aspartate aminotransferases (AATases), except those of animal cytosolic, Ac (His68*:Glu265), and plant mitochondrial, Pm (Met68*:Gln265), origins, was evaluated in the Escherichia coli AATase. Two double-mutant cycles, to K68M/E265Q and the charge reversed K68E/E265K, were characterized with the context dependence (C) and impact (I) formalism, previously defined for functional chimeric analysis. Mutations of Lys68* with Glu265 fixed are generally more deleterious than the converse mutations of Glu265 with Lys68* fixed, showing that buried negative charges have greater effects than buried positive charges in this context. Replacement of the charged Lys68*:Glu265 with the K68M/E265Q neutral pair introduces relatively small effects on the kinetic parameters. The differential sensitivity of k(cat)/K(M, L-Asp) and k(cat)/K(M, alpha-KG) to salt bridge mutagenic replacements is shown by a linear-free energy relationship, in which the logarithms of the latter second order rate constants are generally decreased by a factor of two more than are those of the former. Thus, k(cat)/K(M, L-Asp) and k(cat)/K(M, alpha-KG) are 133 and 442 mM(-1)s(-1) for the wild-type (WT) enzyme, respectively, but their relative order is reversed in the more severely compromised mutants (14.8 and 5.3 mM(-1)s(-1) for K68E). A Venn diagram illustrates apparent forced covariances of groups of amino acids that accompany the naturally occurring salt bridge replacements in the Pm and Ac classes. The more deeply rooted tree indicates that the Csb variant was the ancestral specie.     

Assay for glucose oxidase from Aspergillus niger and Penicillium amagasakiense by Fourier transform infrared spectroscopy

A simple and direct assay method for glucose oxidase (EC 1.1.3.4) from Aspergillus niger and Penicillium amagasakiense was investigated by Fourier transform infrared spectroscopy. This enzyme catalyzed the oxidation of d-glucose at carbon 1 into d-glucono-1,5-lactone and hydrogen peroxide in phosphate buffer in deuterium oxide ((2)H(2)O). The intensity of the d-glucono-1,5-lactone band maximum at 1212 cm(-1) due to CO stretching vibration was measured as a function of time to study the kinetics of d-glucose oxidation. The extinction coefficient epsilon of d-glucono-1,5-lactone was determined to be 1.28 mM(-1)cm(-1). The initial velocity is proportional to the enzyme concentration by using glucose oxidase from both A. niger and P. amagasakiense either as cell-free extracts or as purified enzyme preparations. The kinetic constants (V(max), K(m), k(cat), and k(cat)/K(m)) determined by Lineweaver-Burk plot were 433.78+/-59.87U mg(-1) protein, 10.07+/-1.75 mM, 1095.07+/-151.19s(-1), and 108.74 s(-1)mM(-1), respectively. These data are in agreement with the results obtained by a spectrophotometric method using a linked assay based on horseradish peroxidase in aqueous media: 470.36+/-42.83U mg(-1) protein, 6.47+/-0.85 mM, 1187.77+/-108.16s(-1), and 183.58 s(-1)mM(-1) for V(max), K(m), k(cat), and k(cat)/K(m), respectively. Therefore, this spectroscopic method is highly suited to assay for glucose oxidase activity and its kinetic parameters by using either cell-free extracts or purified enzyme preparations with an additional advantage of performing a real-time measurement of glucose oxidase activity.     

Carbonic anhydrase inhibitors. Inhibition of the beta-class enzyme from the yeast Saccharomyces cerevisiae with anions

The protein encoded by the Nce103 gene of Saccharomyces cerevisiae, a beta-carbonic anhydrase (CA, EC 4.2.1.1) designated as scCA, has been cloned, purified, characterized kinetically, and investigated for its inhibition with a series simple, inorganic anions such as halogenides, pseudohalogenides, bicarbonate, carbonate, nitrate, nitrite, hydrogen sulfide, bisulfite, perchlorate, sulfate, and some of its isosteric species. The enzyme showed high CO(2) hydrase activity, with a k(cat) of 9.4x10(5) s(-1) and k(cat)/K(m) of 9.8x10(7) M(-1) s(-1). scCA was weakly inhibited by metal poisons (cyanide, azide, cyanate, thiocyanate, K(I)s of 16.8-55.6 mM) and strongly inhibited by bromide, iodide, and sulfamide (K(I)s of 8.7-10.8 microM). The other investigated anions showed inhibition constants in the low millimolar range.     

Carbonic anhydrase inhibitors. Cloning, characterization and inhibition studies of the cytosolic isozyme III with anions

The cytosolic human carbonic anhydrase (hCA, EC 4.2.1.1) isozyme III (hCA III) has been cloned and purified by the GST-fusion protein method. Recombinant pure hCA III had the following kinetic parameters for the CO(2) hydration reaction at 20 degrees C and pH 7.5: k(cat) of 1.3 x 10(4) s(- 1) and k(cat)/K(M) of 2.5.10(5) M(- 1) s(- 1). The first detailed inhibition study of this enzyme with anions is reported. Inhibition data of the cytosolic isozymes hCA I - hCA III with a large number of anions (halides, pseudohalides, bicarbonate, carbonate, nitrate, nitrite, hydrosulfide, sulfate, sulfamic acid, sulfamide, etc.), were determined and these values are comparatively discussed for these three cytosolic isoforms. Fluoride, nitrate, nitrite, phenylboronic acid and phenylarsonic acid (as anions) were weak hCA III inhibitors (K(I)s of 21-78.5 mM), whereas bicarbonate, chloride, bromide, sulfate and several other simple anions showed K(I)s around 1 mM. The best hCA III inhibitors were carbonate, cyanide, thiocyanate, azide and hydrogensulfide, which showed K(I)s in the range of 10-90 microM. It is difficult to explain the inhibitory activity of carbonate (K(I) of 10 microM) against hCA III, also considering the fact that this ion has an affinity of 15-73 mM for hCA I and II and is in equilibrium with one of the substrates of this enzyme, i.e., bicarbonate, which is a much weaker inhibitor (K(I) of 0.74 mM against hCA III, of 12 mM against hCA I and of 85 mM against hCA II).     

Human bile salt-dependent lipase efficiency on medium-chain acyl-containing substrates: control by sodium taurocholate

Bile salt-dependent lipase was purified to homogeneity from lyophilized human milk and used to screen the influence of the acyl chain length (2-16 carbon atoms) on the kinetic constants k(cat) and K(m) of the hydrolysis of para-nitrophenyl (pnp) ester substrates in the presence or absence of sodium taurocholate (NaTC: 0.02-20 mM). The highest k(cat) value (～3,500 s(-1)) was obtained with pnpC(8) as substrate, whereas the lowest K(m) (<10 μM) was that recorded with pnpC(10). In the absence of NaTC, the maximal catalytic efficiency (k(cat)/K(m)) was obtained with pnpC(8), while in the presence of NaTC k(cat)/K(m) was maximal with pnpC(8), pnpC(10) or pnpC(12). The bile salt activated the enzyme in two successive saturation phases occurring at a micromolar and a millimolar concentration range, respectively. The present data emphasize the suitability of this enzyme for the hydrolysis of medium-chain acyl-containing substrates and throw additional light on how BSDL is activated by NaTC.     

Synthesis and hydrolysis by cysteine and serine proteases of short internally quenched fluorogenic peptides

We developed sensitive substrates for cysteine proteases and specific substrates for serine proteases based on short internally quenched fluorescent peptides, Abz-F-R-X-EDDnp, where Abz (ortho-aminobenzoic acid) is the fluorescent donor, EDDnp [N-(ethylenediamine)-2,4-dinitrophenyl amide] is the fluorescent quencher, and X are natural amino acids. This series of peptides is compared to the commercially available Z-F-R-MCA, where Abz and X replace carbobenzoxy (Z) and methyl-7-aminocoumarin amide (MCA), respectively; and EDDnp can be considered a P(2)' residue. Whereas MCA is the fluorescent probe and cannot be modified, in the series Abz-F-R-X-EDDnp the amino acids X give the choice of matching the specificity of the S(1)' enzyme subsite, increasing the substrate specificity for a particular protease. All Abz-F-R-X-EDDnp synthesized peptides (for X = Phe, Leu, Ile, Ala, Pro, Gln, Ser, Lys, and Arg) were assayed with papain, human cathepsin L and B, trypsin, human plasma, and tissue kallikrein. Abz-F-R-L-EDDnp was the best substrate for papain and Abz-F-R-R-EDDnp or Abz-F-R-A-EDDnp was the more susceptible to cathepsin L. Abz-F-R-L-EDDnp was able to detect papain in the range of 1 to 15 pM. Human plasma kallikrein hydrolyzed Abz-F-R-R-EDDnp with significant efficiency (k(cat)/K(m) = 1833 mM(-1) s(-1)) and tissue kallikrein was very selective, hydrolyzing only the peptides Abz-F-R-A-EDDnp (k(cat)/K(m) = 2852 mM(-1) s(-1)) and Abz-F-R-S-EDDnp (k(cat)/K(m) = 4643 mM(-1) s(-1)). All Abz-F-R-X-EDDnp peptides were resistant to hydrolysis by thrombin and activated factor X.     

High-level expression of human liver monoamine oxidase B in Pichia pastoris

The high-level heterologous expression, purification, and characterization of the mitochondrial outer membrane enzyme human liver monoamine oxidase B (MAO B) using the methylotrophic yeast Pichia pastoris expression system are described. A 2-L culture of P. pastoris expresses approximately 1700 U of MAO B activity, with the recombinant enzyme associated tightly with the membrane fraction of the cell lysate. By a modification of the published procedure for purification of bovine liver MAO B [Salach, J. I. (1979) Arch. Biochem. Biophys. 192, 128-137], recombinant human liver MAO B is purified in a 34% yield ( approximately 200 mg from 2 L of cell culture). The isolated enzyme exhibits an M(r) of approximately 60, 000 on SDS-PAGE and 59,474 from electrospray mass spectrometry measurements, which is in good agreement with the mass predicted from the gene sequence and inclusion of the covalent FAD. One mole of covalent FAD per mole of MAO B is present in the purified enzyme and is bound by an 8alpha-S-cysteinyl(397) linkage, as identified by electrospray mass spectrometry of the isolated tryptic/chymotryptic flavin peptide. Recombinant human liver MAO B and bovine liver MAO B are shown to be acetylated at the seryl residues at their respective amino termini. The benzylamine oxidase activity of recombinant MAO B ranges from 3.0 to 3.4 U/mg and steady-state kinetic parameters for this enzyme preparation compare well with those published for the bovine liver enzyme: k(cat) = 600 min(-1), K(m)(benzylamine) = 0.50 mM, and K(m)(O(2)) = 0.33 mM. Kinetic isotope effect parameters using [alpha,alpha-(2)H(2)]benzylamine are also similar to those found for the bovine enzyme. Recombinant MAO B exhibits a (D)k(cat) = 4.7, a (D)[k(cat)/K(m)(benzylamine)] = 4.5, and a (D)[k(cat)/K(m)(O(2))] = 1.0. In contrast to bovine liver MAO B, no evidence was found for the presence of any anionic flavin radical either by UV-vis or by EPR spectroscopy in the resting form of the enzyme. These data demonstrate the successful heterologous expression of a functional, membrane-bound MAO B, which will permit a number of mutagenesis studies as structural and mechanistic probes not previously possible.     

Catalytic properties of the PepQ prolidase from Escherichia coli

The PepQ prolidase from Escherichia coli catalyzes the hydrolysis of dipeptide substrates with a proline residue at the C-terminus. The pepQ gene has been cloned, overexpressed, and the enzyme purified to homogeneity. The k(cat) and k(cat)/K(m) values for the hydrolysis of Met-Pro are 109 s(-1) and 8.4 x 10(5)M(-1)s(-1), respectively. The enzyme also catalyzes the stereoselective hydrolysis of organophosphate triesters and organophosphonate diesters. A series of 16 organophosphate triesters with a p-nitrophenyl leaving group were assessed as substrates for PepQ. The S(P)-enantiomer of methyl phenyl p-nitrophenyl phosphate was hydrolyzed with a k(cat) of 36 min(-1) and a k(cat)/K(m) of 710 M(-1)s(-1). The corresponding R(P)-enantiomer was hydrolyzed more slowly with a k(cat) of 0.4 min(-1) and a k(cat)/K(m) of 11 M(-1)s(-1). The PepQ prolidase can be utilized for the kinetic resolution of racemic phosphate esters. The PepQ prolidase was shown to hydrolyze the p-nitrophenyl analogs of the nerve agents GB (sarin), GD (soman), GF, and VX.     

Nardilysin cleaves peptides at monobasic sites

Nardilysin (N-arginine dibasic convertase, EC 3.4.24.61) was first identified on the basis of its ability to cleave peptides containing an arginine dibasic pair, i.e., Arg-Arg or Arg-Lys. However, it was observed that an aromatic residue adjacent to the dibasic pair (i.e., Phe-Arg-Lys) could alter the cleavage site. In this study we determined whether nardilysin can cleave peptides at a single basic residue. Nardilysin cleaves beta-endorphin at the monobasic site, Phe(17)-Lys(18), with a k(cat)/K(m) of 2 x 10(8) M(-)(1) min(-)(1). This can be compared to a k(cat)/K(m) of 8.5 x 10(8) M(-)(1) min(-)(1) for cleavage between a dibasic pair in dynorphin B-13. Nardilysin also cleaves calcitonin at His-Arg and somatostatin-14 at Cys-Lys. We examined the hydrolysis of fluorogenic peptides based on the beta-endorphin 12-24 sequence, Abz-T-P-L-V-T-L-X(1)-X(2)-N-A-I-I-K-Q-EDDnp. Nardilysin hydrolyzes the peptides when X(1)-X(2) = F-K, F-R, W-K, M-K, Y-K, and L-K. The kinetics of cleavage at F-K and F-R are similar; however, K-F is not hydrolyzed. Nardilysin cleaves at two monobasic sites M-K and F-R of the kallidin model peptide Abz-MISLMKRPPGFSPFRSSRI-NH(2), releasing desArg(10) kallidin (KRPPGFSPF). However, nardilysin does not release desArg(10) kallidin from the physiological precursor low molecular weight kininogen. These studies extend the range of potential substrates for nardilysin and further substantiate that nardilysin is a true peptidase.     

Carbonic anhydrase inhibitors. Inhibition studies of the human secretory isoform VI with anions

The unique secretory isozyme of human carbonic anhydrase (hCA, EC 4.2.1.1), hCA VI, has been cloned, expressed, and purified. The kinetic parameters for the CO(2) hydration reaction proved hCA VI to possess a k(cat) of 3.4x10(5)s(-1) and k(cat)/K(M) of 4.9x10(7)M(-1)s(-1) (at pH 7.5 and 20 degrees C). hCA VI has a significant catalytic activity for the physiological reaction, of the same order of magnitude as isoforms CA I or CA IX. A series of anions (such as bicarbonate, chloride, nitrate, etc.) were shown to inhibit the activity of the enzyme, with inhibition constants typically in the range of 0.60-0.90mM. The best hCA VI inhibitors were cyanide, azide, sulfamide, and sulfamate, with inhibition constants in the range of 70-90microM.     

Designing of substrates and inhibitors of bovine alpha-chymotrypsin with synthetic phenylalanine analogues in position P(1)

The primary specificity residue of a substrate or an inhibitor, called the P(1) residue, is responsible for the proper recognition by the cognate enzyme. This residue enters the S(1) pocket of the enzyme and establishes contacts (up to 50%) inside the proteinase substrate cavity, strongly affecting its specificity. To analyze the influence on bovine alpha-chymotrypsin substrate activity, aromatic non-proteinogenic amino acid residues in position P(1) with the sequence Ac-Phe-Ala-Thr-X-Anb(5,2)-NH(2) were introduced: L-pyridyl alanine (Pal), 4-nitrophenylalanine - Phe(p-NO(2)), 4-aminophenylalanine - Phe(p-NH(2)), 4-carboxyphenylalanine Phe(p-COOH), 4-guanidine phenylalanine - Phe(p-guanidine), 4-methyloxycarbonyl-phenylalanine - Phe(p-COOMe), 4-cyanophenylalanine - Phe(p-CN), Phe, Tyr. The effect of the additional substituent at the phenyl ring of the Phe residue was investigated. All peptides contained an amide of 5-amino-2-nitrobenzoic acid, which served as a chromophore. Kinetic parameters (k(cat), K(M) and k(cat)/K(M)) of the peptides synthesized with bovine alpha-chymotrypsin were determined. The highest value of the specificity constant k(cat)/K(M), reaching 6.0 x 10(5) [M(-1)xs(-1)], was obtained for Ac-Phe-Ala-Thr-Phe(p-NO(2))-Anb(5,2)-NH(2). The replacement of the acetyl group with benzyloxycarbonyl moiety yielded a substrate with the value of k(cat) more than three times higher. Peptide aldehydes were synthesized with selected residues (Phe, Pal, Tyr, Phe(p-NO(2)) in position P(1) and potent chymotrypsin inhibitors were obtained. The dissociation constant (K(i)) with the experimental enzyme determined for the most active peptide, Tos-Phe-Ala-Thr-Phe(p-NO(2))-CHO, amounted to 1.12 x 10(-8) M.     

Functional characterization of a novel ArgA from Mycobacterium tuberculosis

The Mycobacterium tuberculosis gene Rv2747 encodes a novel 19-kDa ArgA that catalyzes the initial step in L-arginine biosynthesis, namely the conversion of L-glutamate to alpha-N-acetyl-L-glutamate. Initial velocity studies reveal that Rv2747 proceeds through a sequential kinetic mechanism, with K(m) values of 280 mM for L-glutamine and 150 microM for acetyl-coenzyme A and with a k(cat) value of 200 min(-1). Initial velocity studies with L-glutamate showed that even at concentrations of 600 mM, saturation was not observed. Therefore, only a k(cat)/K(m) value of 125 M(-1) min(-1) can be calculated. Inhibition studies reveal that the enzyme is strongly regulated by L-arginine, the end product of the pathway (50% inhibitory concentration, 26 microM). The enzyme was completely inhibited by 500 microM arginine, with a Hill coefficient of 0.60, indicating negatively cooperative binding of L-arginine.