Purification of biotinidase from human plasma and its activity on biotinyl peptides

Biotinidase catalyzes the hydrolysis of N epsilon-biotinyllysine (biocytin) to form biotin and free lysine. The enzyme has been purified 4800-fold from outdated human plasma and was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to have a molecular weight of (76 +/- 2) X 10(3). The same molecular weight was found by molecular sieve chromatography under nondenaturing conditions, indicating biotinidase is a monomer. This value is in contrast to a molecular weight of 115 000 determined by Pispa [Pispa, J. (1965) Ann. Med. Exp. Biol. Fenn., Suppl. 5, 5-39] with an impure biotinidase. The Km for biocytin was 6.2 X 10(-6) M, and biotinidase was found to be sensitive to phenylmethanesulfonamide and iodoacetamide in agreement with earlier studies by Knappe and co-workers [Knappe, J., Brümmer, W., & Bierderbick, K. (1963) Biochem. Z. 338, 599-613], who suggested that serine hydroxyl groups and sulfhydryl groups are essential for enzymatic activity. The specificity of biotinidase was examined by using synthetic and natural biotinyl peptides isolated by specific proteolytic cleavage of the biotinyl subunit of transcarboxylase. It was found that the rate of hydrolysis of biocytin was 83-fold higher than that found for biotin-containing peptides 5-13 residues in length. Removal of methionine from either side of the conserved region around the biocytin did not greatly alter the rate of cleavage. Increasing the peptide to 65-123 residues in length decreased the rate 1200-fold compared to that of biocytin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enkephalin-degrading aminopeptidase in the longitudinal muscle layer of guinea pig small intestine: its properties and action on neuropeptides.

A membrane-bound enkephalin-degrading aminopeptidase was purified from the longitudinal muscle layer of the guinea pig small intestine by four steps of column chromatography using L-tyrosine beta-naphthylamide. The molecular weight of the enzyme was estimated to be 105,000 by gel filtration. The maximum activity was observed between pH 6.5 and 7.0. The Km value for leucine-enkephalin was 137 microM. The aminopeptidase activity toward aminoacyl beta-naphthylamide substrates was restricted to basic, neutral, and aromatic aminoacyl derivatives. No action was detected on acidic amino acid and proline derivatives. The enzyme was potently inhibited by the aminopeptidase inhibitors actinonin, amastatin, and bestatin, and bioactive peptides such as angiotensin III, substance P, and Met-Lys-bradykinin. The enzyme activity was also inhibited by the antibody against the purified serum enkephalin-degrading aminopeptidase of guinea pig at concentrations similar to those at which activity was observed toward serum enkephalin-degrading aminopeptidase and renal aminopeptidase M. The enzyme rapidly hydrolyzed Leu-enkephalin and Met-enkephalin with the sequential removal of the N-terminal amino acid residues. The enzyme also hydrolyzed two enkephalin derivatives, angiotensin III and neurokinin A. However, neurotensin, substance P, and bradykinin were not cleaved. These properties indicated that the membrane-bound enkephalin-degrading aminopeptidase in the longitudinal muscle layer of the small intestine is similar to the serum enkephalin-degrading aminopeptidase and resembles aminopeptidase M. It is therefore suggested to play an important role in the metabolism of some bioactive peptides including enkephalin in peripheral nervous systems in vivo.     

Human adenovirus proteinase: DNA binding and stimulation of proteinase activity by DNA.

The interaction of the human adenovirus proteinase (AVP) with various DNAs was characterized. AVP requires two cofactors for maximal activity, the 11-amino acid residue peptide from the C-terminus of adenovirus precursor protein pVI (pVIc) and the viral DNA. DNA binding was monitored by changes in enzyme activity or by fluorescence anisotropy. The equilibrium dissociation constants for the binding of AVP and AVP-pVIc complexes to 12-mer double-stranded (ds) DNA were 63 and 2.9 nM, respectively. DNA binding was not sequence specific; the stoichiometry of binding was proportional to the length of the DNA. Three molecules of the AVP-pVIc complex bound to 18-mer dsDNA and six molecules to 36-mer dsDNA. When AVP-pVIc complexes bound to 12-mer dsDNA, two sodium ions were displaced from the DNA. A Delta of -4.6 kcal for the nonelectrostatic free energy of binding indicated that a substantial component of the binding free energy results from nonspecific interactions between the AVP-pVIc complex and DNA. The cofactors altered the interaction of the enzyme with the fluorogenic substrate (Leu-Arg-Gly-Gly-NH)2-rhodamine. In the absence of any cofactor, the Km was 94.8 microM and the kcat was 0.002 s(-1). In the presence of adenovirus DNA, the Km decreased 10-fold and the kcat increased 11-fold. In the presence of pVIc, the Km decreased 10-fold and the kcat increased 118-fold. With both cofactors present, the kcat/Km ratio increased 34000-fold, compared to that with AVP alone. Binding to DNA was coincident with stimulation of proteinase activity by DNA. Although other proteinases have been shown to bind to DNA, stimulation of proteinase activity by DNA is unprecedented. A model is presented suggesting that AVP moves along the viral DNA looking for precursor protein cleavage sites much like RNA polymerase moves along DNA looking for a promoter.     

Determination of biocytin

Biocytin (epsilon-N-[d-biotinyl]-L-lysine) is generally undetected in serum and other body fluids of normal healthy individuals in view of the ubiquitous distribution of biotinidase. It has been suggested that biocytin may be present in serum and urine of patients with inherited biotinidase deficiency. We have developed a noncompetitive assay for biocytin based on its interaction with avidin. Biocytin can be determined in biological samples containing both biotin and biocytin. Biotin from such samples is removed by an anion-exchange resin and the biocytin is determined by the avidin-binding assay. The effect of above-normal levels of biotin in the sample on the assay of biocytin is discussed.     

Use of a fluorescence plate reader for measuring kinetic parameters with inner filter effect correction

A general method is presented here for the determination of the Km, kcat, and kcat/Km of fluorescence resonance energy transfer (FRET) substrates using a fluorescence plate reader. A simple empirical method for correcting for the inner filter effect is shown to enable accurate and undistorted measurements of these very important kinetic parameters. Inner filter effect corrected rates of hydrolysis of a FRET peptide substrate by hepatitis C virus (HCV) NS3 protease at various substrate concentrations enabled measurement of a Km value of 4.4 +/- 0.3 microM and kcat/Km value of 96,500 +/- 5800 M-1 s-1. These values are very close to the HPLC-determined Km value of 4.6 +/- 0.7 microM and kcat/Km value of 92,600 +/- 14,000 M-1 s-1. We demonstrate that the inner filter effect correction of microtiter plate reader velocities enables rapid measurement of Ki and Ki' values and kinetic inhibition mechanisms for HCV NS3 protease inhibitors.     

Replacement of an interdomain residue Val39 of Escherichia coli aspartate aminotransferase affects the catalytic competence without altering the substrate specificity of the enzyme

Three mutant Escherichia coli aspartate aminotransferases in which Val39 was changed to Ala, Leu, and Phe by site-directed mutagenesis were prepared and characterized. Among the three mutant and the wild-type enzymes, the Leu39 enzyme had the lowest Km values for dicarboxylic substrates. The Km values of the Ala39 enzyme for dicarboxylates were essentially the same as those of the wild-type (Val39) enzyme. These two mutant enzymes showed essentially the same kcat values for dicarboxylic substrates as did the wild-type enzyme. On the other hand, incorporation of a bulky side-chain at position 39 (Phe39 enzyme) decreased both the affinity (1/Km) and catalytic ability (kcat) toward dicarboxylic substrates. These results show that the position 39 residue is involved in the modulation of both the binding of dicarboxylic substrates to enzyme and the catalytic ability of the enzyme. Although the replacement of Val39 with other residues altered both the kcat and Km values toward various substrates including dicarboxylic and aromatic amino acids and the corresponding oxo acids, it did not alter the ratio of the kcat/Km value of the enzyme toward a dicarboxylic substrate to that for an aromatic substrate. The affinity for aromatic substrates was not affected by changing the residue at position 39. These data indicate that, although the side chain bulkiness of the residue at position 39 correlates well with the activity toward aromatic substrates in the sequence alignment of several aminotransferases [Seville, M., Vincent M.G., & Hahn, K. (1988) Biochemistry 27, 8344-8349], the residue does not seem to be involved in the recognition of aromatic substrates.     

Effectors of HIV-1 protease peptidolytic activity

High concentrations of salts dramatically affect the interaction of small ligands with HIV-1 protease. For instance, the Km and kcat values for Abz-Thr-Ile-Nle-p-nitro-Phe-Gln-Arg-NH2 (S) increased 120-fold and 3-fold, respectively, as the NaCl concentration in the assay decreased from 4.0 to 0.5 M. The Kd value for the competitive inhibitor amprenavir increased 12-fold over this concentration range of NaCl. The bimolecular rate constant for association of enzyme with amprenavir was independent of NaCl concentration, whereas the dissociation rate constant decreased with increasing NaCl concentration. Polyanionic polymers such as heparin or poly A substituted for NaCl. For example, the value of kcat/Km for S was 0.18 microM(-1) x s(-1) when the enzyme (<10 nM) was assayed in the standard buffer supplemented with 5 mM NaCl. If 0.01% poly A were included, the value of kcat/Km increased to 8.6 microM(-1) x s(-1). A DNA oligomer (23-mer) with an hexachlorofluoresceinyl moiety linked to the 5' end was studied as a model polyanionic polymer. The enzyme bound HF23 (Kd < 1 nM) with concomitant quenching of the hexachlorofluoresceinyl fluorescence. The stoichiometry for binding was 3 mol of enzyme per mol of oligomer. The hydrolytic activity of the enzyme with this oligomer was similar to that observed with poly A or high salt concentration when the molar ratio of oligomer to enzyme was greater than one. The results presented herein demonstrate that polyanionic polymers substitute for salts as effectors of HIV protease.     

ES complexes of Aeromonas aminopeptidase: direct observation by stopped-flow fluorescence

Intermediates of Aeromonas aminopeptidase are monitored through fluorescence generated by radiationless energy transfer (RET) between enzyme tryptophans and the dansyl group of the bound substrate. Upon binding of the substrate enzyme tryptophan fluorescence is quenched and substrate dansyl fluorescence enhanced. These processes are reversed upon hydrolysis of the Leu-Ala bond and release of Ala-DED from the enzyme. Stopped-flow RET kinetic analysis yields values of kcat = 36 sec-1 and Km = 3.7 microM at pH 7.5 and 20 degrees C. These values represent the highest kcat/Km ratio, 1 X 10(7) M-1 sec-1, of any substrate for Aeromonas aminopeptidase. The excellent binding properties of the peptide permit direct visualization of ES complexes even at enzyme concentrations of 10(-7) M.     

Modified activity of Aeromonas aminopeptidase: metal ion substitutions and role of substrates

Aeromonas aminopeptidase contains two nonidentical metal binding sites that have been shown by both spectroscopy and kinetics to be capable of interacting with one another [Prescott, J.M., Wagner, F.W., Holmquist, B., & Vallee, B.L. (1985) Biochemistry 24, 5350-5356]. The effects of metal ion substitutions on the susceptibility of the p-nitroanilides of L-alanine, L-valine, and L-leucine--substrates that are hydrolyzed at widely differing rates by native Aeromonas aminopeptidase--were studied by determining values of kcat and Km for the 16 metalloenzymes that result from all possible combinations of Zn2+, Co2+, Ni2+, and Cu2+ in each of the two sites. The different combinations of metal ions and substrates yield a broad range in kinetic values; kcat varies by more than 1800-fold, Km by 3000-fold, and kcat/Km ratios by more than 10,000. L-Leucine-p-nitroanilide is by far the most susceptible of the three substrates, and the hyperactivation previously observed with aminopeptidase containing either Ni2+ or Cu2+ in the first binding site and Zn2+ in the second site occurs only with the two poorer substrates, L-alanine-p-nitroanilide and L-valine-p-nitroanilide. Although the enzyme with Zn2+ in both sites hydrolyzes the substrates with N-terminal alanine and valine poorly, it is extremely effective toward L-leucine-p-nitroanilide. Neither metal binding site can be identified as controlling either Km or kcat; both parameters are influenced by the identity of the metal ions, by the site each occupies, and, most strongly, by the substrate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparative enzymatic studies of human renin acting on pure natural or synthetic substrates

Some of the essential structural requirements for the enzymatic reaction of pure human renin acting on pure human and rat angiotensinogen and on their synthetic tetradecapeptide substrates were investigated. The five carboxy terminal amino acids of synthetic tetradecapeptides played a significant role in substrate recognition and/or hydrolysis by human renin. Kinetic constants Km, Kcat and kcat/Km of the various human renin assays were different according to the substrate used. The presence of either an asparagine or a threonine residue in the S'4 renin subsite did not affect significantly the kinetic constant values. A tyrosine residue, rather than a histidine residue, in the S'3 renin subsite gave the best synthetic substrate studied. When tyrosine residue was present in the S'2 renin subsite an important decrease in kcat was observed. Human angiotensinogen was hydrolysed by human renin with lower Km and kcat values than those measured with human and porcine synthetic substrates, suggesting that the 3-dimensional structure of human angiotensinogen plays a key role in the hydrolysis. This finding was supported by assays performed with rat angiotensinogen, which was cleared by human renin with the same kcat value as rat tetradecapeptide, but with a 49-fold lower Km. Between human and rat angiotensinogen a kcat/Km value of only 2-fold higher has been found in the renin assay using human substrate.     

Kinetic studies of wheat carboxypeptidase-catalyzed reaction: differences in pressure and temperature dependence of peptidase and esterase activities

A kinetic study of hydrolytic catalysis by wheat bran carboxypeptidase (carboxypeptidase W) was carried out using 3-(2-furyl)acryloyl-acylated (Fua-) synthetic substrates. This enzyme showed high esterase activity in addition to the intrinsic carboxypeptidase activity. The optimum pH for the peptidase activity (kcat/Km) was at pH 3.3 and the kcat/Km value decreased with increasing pH with an apparent pKa of 4.50, while the esterase activity increased with pH up to pH 8 with an apparent pKa of 6.04. Optimum pH's for kcat for the peptidase and esterase reactions were also very different and their apparent pKa values were 3.80 and 6.15, respectively. From a measurement of the pressure dependences of kcat and Km, the activation volumes (delta V not equal to) and reaction volumes (delta V), respectively, were determined. delta V not equal to for kcat was -7 to -8 ml/mol for peptidase and -2 to -3 ml/mol for esterase. These results lead us to propose that the peptidase and esterase activities of carboxypeptidase W are different not in the rate-determining steps in a common reaction pathway, but in the binding modes and/or catalytic site(s).     

Purification and characterization of human serum biotinidase

Biotinidase has been purified from human serum to a specific activity of 1900 units/mg protein by a five-step procedure. After ammonium sulfate precipitation (33-55% cut) it was purified by DEAE-Sephacel, hydroxylapatite, octyl-Sepharose CL-4B, and Sephadex G-100 chromatography. The purified enzyme showed a single silver staining band with polyacrylamide gel electrophoresis under denaturing and non-denaturing conditions. Biotinidase is a glycoprotein. The sialic acid residues in the molecule are not required for enzyme activity. The Mr of human serum biotinidase estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Ferguson plot) and by sedimentation analysis was 68,000. Human serum biotinidase showed maximum activity in the pH range 6.0 to 7.5 with N-(d-biotinyl) p-aminobenzoate as substrate. However, with biocytin as substrate, the maximal activity of the enzyme was in the pH range 4.5 to 6.0. Using structural analogs of the substrate we have shown that biotinidase is not a general proteolytic enzyme and has specific structural requirements in the substrate for hydrolysis.     

Human cathepsin H: deletion of the mini-chain switches substrate specificity from aminopeptidase to endopeptidase

The mini-chain of human cathepsin H has been identified as the major structural element determining the protease's substrate specificity. A genetically engineered mutant of human cathepsin H lacking the mini-chain, des[Glu(-18)-Thr(-11)]-cathepsin H, exhibits endopeptidase activity towards the synthetic substrate Z-Phe-Arg-NH-Mec (kcat = 0.4 s(-1), Km = 92 microM, kcat/Km = 4348 M(-1) s(-1)) which is not cleaved by r-wt cathepsin H. However, the mutant enzyme shows only minimal aminopeptidase activity for H-Arg-NH-Mec (kcat = 0.8 s(-1), Km = 3.6 mM, kcat/Km = 222 M(-1) s(-1)) which is one of the best known substrates for native human cathepsin H (kcat = 2.5 s(-1), Km = 150 microM, kcat/Km = 16666 M(-1) s(-1)). Inhibition studies with chicken egg white cystatin and E-64 suggest that the mini-chain normally restricts access of inhibitors to the active site. The kinetic data on substrates hydrolysis and enzyme inhibition point out the role of the mini-chain as a structural framework for transition state stabilization of free alpha-amino groups of substrates and as a structural barrier for endopeptidase-like substrate cleavage.     

Lyophyllum cinerascens aminopeptidase: purification and enzymatic properties

An aminopeptidase (EC 3.4.11.1) was purified from the extract of Lyophyllum cinerascens by ammonium sulfate fractionation and sequential chromatographies on DEAE-Sephadex, Sephadex G-150, HPLC-phenyl-5PW, and HPLC-DEAE-5PW columns, with an activity recovery of 4.6% using Leu-beta-naphthylamide as a substrate. The enzyme was a tetrameric protein of molecular weight 150,000 and was found to be rich in histidine. It exhibited a pH optimum of 7.2 and stability between pH 5.7 and 7.7. The isoelectric point of the enzyme was 4.6. The enzyme catalyzed the hydrolysis of amino acid beta-naphthylamides, Phe greater than Leu greater than Met greater than Tyr greater than Ala greater than Glu, and the differences of the measured kcat's ranged over 2-3 orders of magnitude while many of the amino acid beta-naphthylamides were not hydrolyzed at all. Other interesting comparisons include two aliphatics, Ala vs Leu, and the aromatics, Tyr vs Phe, which show a 30-fold difference in the kcat/Km values. The enzyme also hydrolyzed Leu-Gly-Gly and the B chain of oxidized insulin to release N-terminal leucine and phenylalanine, respectively. The release of N-terminal Phe from the oxidized B chain is interesting in view of the fact that the penultimate residue is Val, an unfavorable amino acid in the beta-naphthylamide series. The enzyme seems to be a true aminopeptidase, requiring the free amino groups and hydrolyzing dipeptide and oligopeptide from the N-terminal end. The enzyme was resistant to the action of amastatin. Neither sulfhydryl reagents nor serine protease inhibitors affected the enzyme activity; however, the enzyme was inhibited weakly by EDTA and bestatin and strongly by diethyl pyrocarbonate.     

A cold-adapted epoxide hydrolase from a strict marine bacterium, Sphingophyxis alaskensis

An open reading frame (ORF) encoding a putative epoxide hydrolase (EHase) was identified by analyzing the genome sequence of Sphingophyxis alaskensis. The EHase gene (seh) was cloned and expressed in E. coli. To facilitate purification, the gene was fused in-frame to 6x histidine at the C-terminus. The recombinant EHase (rSEH) was highly soluble and could be purified to apparent homogeneity by one step of metal affinity chromatography. The purified SEH displayed hydrolyzing activities toward various epoxides such as styrene oxide, glycidyl phenyl ether, epoxyhexane, epoxybutane, epichlorohydrin, and epifluorohydrin. The optimum activity toward styrene oxide was observed at pH 6.5 and 35 degrees . The purified SEH showed a coldadapted property, displaying more than 40% of activity at low temperature of 10 degrees compared with the optimum activity. Despite the catalytic efficiency, the purified SEH did not hydrolyze various epoxides enantioselectively. Km and kcat of SEH toward (R)-styrene oxide were calculated as 4+/-0.3 mM and 7.42 s-1, respectively, whereas Km and kcat of SEH toward (S)-styrene oxide were 5.25+/-0.3 mM and 10.08 s-1, respectively.     

Mechanistic studies on thrombin catalysis

The kinetic mechanism of the cleavage of four p-nitroanilide (pNA) substrates by human alpha-thrombin has been investigated by using a number of steady-state kinetic techniques. Solvent isotope and viscosity effects were used to determine the stickiness of the substrates at the pH optimum of the reaction; a sticky substrate is defined as one that undergoes catalysis faster than it dissociates from the Michaelis complex. Whereas benzoyl-Arg-pNA could be classified as a nonsticky substrate, D-Phe-pipecolyl-Arg-pNA was very sticky. The other two substrates (tosyl-Gly-Pro-Arg-pNA and acetyl-D-Phe-pipecolyl-Arg-pNA) were slightly sticky. The pH profiles of kcat/Km were bell-shaped for all substrates. The pKa values determined from the pH dependence of kcat/Km for benzoyl-Arg-pNA were about 7.5 and 9.1. Similar pKa values were determined from the pH profiles of kcat/Km for tosyl-Gly-Pro-Arg-pNA and acetyl-D-Phe-pipecolyl-Arg-pNA and for the binding of the competitive inhibitor N alpha-dansyl-L-arginine-4-methylpiperidine amide. The groups responsible for the observed pKa values were proposed to be His57 and the alpha-amino group of Ile16. The temperature dependence of the pKa values was consistent with this assignment. The pKa values of 6.7 and 8.6 observed in the pH profile of kcat/Km for D-Phe-pipecolyl-Arg-pNA were displaced to lower values than those observed for the other substrates. The displacement of the acidic pKa value could be attributed to the stickiness of this substrate.(ABSTRACT TRUNCATED AT 250 WORDS)     

The ywad gene from Bacillus subtilis encodes a double-zinc aminopeptidase

The yet uncharacterized ywad gene from Bacillus subtilis has been cloned and overexpressed in Escherichia coli. The gene product (BSAP) was purified and shown to be an aminopeptidase. The activity of BSAP was optimal at pH 8.4, the enzyme was stable for 20 min at 80 degrees C and its activity was not affected by serine protease and aspartic protease inhibitors, but was completely diminished by the Zn-chelator 1,10-phenanthroline. ZnCl2 was able to restore activity, and the binding stoichiometry of zinc to apo-BSAP indicated two Zn ions per protein molecule. BSAP exhibited high preference toward p-nitroanilide derived Arg, Lys, and Leu synthetic substrates resulting in kcat/Km values of 1-5 x 10(1) s(-1) mM(-1).     

A fluorometric assay for biotinidase

An assay for biotinidase using biocytin, the natural substrate, is described. The fluorometric procedure uses 1,2-diacetylbenzene which reacts selectively with lysine allowing its direct determination in mixture with biocytin. We have examined the applicability of the assay using human serum biotinidase.     

Studies on the aminopeptidase activity of rat cathepsin H.

Three synthetic substrates H-Arg-NH-Mec, Bz-Arg-NH-Mec and H-Cit-NH-Mec (Bz, Benzoyl; NH-Mec, 4-methylcoumaryl-7-amide; Cit, citrulline) were used to characterize specificity requirements for the P1-S1 interaction of cathepsin H from rat liver. From rapid equilibrium kinetic studies it was shown that Km, kcat and the specificity constants kcat/Km are quite similar for substrates with a free alpha-amino group. In contrast, a 25-fold decrease of kcat/Km was observed for the N-terminal-blocked substrate Bz-Arg-NH-Mec. The activation energies for H-Arg-NH-Mec and Bz-Arg-NH-Mec were determined to be 37 kJ/mol and 55 kJ/mol, respectively, and the incremental binding energy delta delta Gb of the charged alpha-amino group was estimated to -8.1 kJ/mol at pH 6.8. The shown preference of cathepsin H for the unblocked substrates H-Arg-NH-Mec and H-Cit-NH-Mec was further investigated by inspection of the pH dependence of kcat/Km. The curves of the two substrates with a charged alpha-amino group showed identical bell-shaped profiles which both exhibit pKa1 and pKa2 values of 5.5 and 7.4, respectively, at 30 degrees C. The residue with a pKa1 of 5.5 in the acid limb of the activity profile of H-Arg-NH-Mec was identified by its ionization enthalpy delta Hion = 21 kJ/mol as a beta-carboxylate or gamma-carboxylate of the enzyme, whereas the residue with a pKa2 of 7.4 was assigned to the free alpha-amino group of the substrate with a delta Hion of 59 kJ/mol. Bz-Arg-NH-Mec showed a different pH-activity profile with a pKa1 of 5.4 and a pKa2 of 6.6 at 30 degrees C. Cathepsin H exhibits no preference for a basic P1 side chain as has been shown by the similar kinetics of H-Arg-NH-Mec and the uncharged, isosteric substrate H-Cit-NH-Mec. In summary, specific interactions of an anionic cathepsin H active site residue with the charged alpha-amino group of substrates caused transition state stabilization which proves the enzyme to act preferentially as an aminopeptidase.     

Purification and characterization of aminopeptidase B from Escherichia coli K-12

Aminopeptidase B, which is one of the four cysteinylglycinases of Escherichia coli K-12, was purified to electrophoretic homogeneity and its enzymatic characteristics were observed. Aminopeptidase B was activated by various divalent cations such as Ni2+, Mn2+, Co2+, and Cd2+, and lost its activity completely on dialysis against EDTA. This indicates that aminopeptidsase B is a metallopeptidase. It was stabilized against heat in the presence of Mn2+ or Co2+. The activity of aminopeptidase B, which was saturated with one of above divalent cations, was enhanced on the addition of a very small amount of a second divalent cation. Alpha-glutamyl p-nitroanilide, leucine p-nitroanilide, and methionine p-nitroanilide were good substrates for aminopeptidase B, while native peptides, cysteinylglycine and leucylglycine, were far better substrates. The kcat/Km for cysteinylglycine was much bigger than those for leucylglycine or leucine p-nitroanilide.