Conformational properties of tripeptides having cyclic dipeptide backbone and their catalytic properties for enantiomer-selective hydrolysis

Conformation in aqueous solution at pH 6.95 of tripeptides having cyclic dipeptide backbones, cyclo[l-Glu(l-Leu-OBzl)-l-His] and cyclo[l-Glu(l-Leu-OH)-l-His], was investigated by u.v., c.d. and n.m.r. spectroscopy and by the lanthanide probe method. In the major conformation of cyclo[l-Glu(l-Leu-OBzl)-l-His], the cyclic dipeptide backbone takes a flagpole-boat conformation in which the sidechain of the l-His residue is nearly parallel with the backbone plane and the sidechain of the l-Glu residue protrudes outside the backbone plane. In the major conformation of cyclo[l-Glu(l-Leu-OH)-l-His], the cyclic dipeptide backbone takes a flagpole-boat conformation in which the sidechains of the l-His and l-Glu residues are accommodated in the same side of the backbone plane so that the imidazolyl sidechain of l-His residue is twisted slightly. Tripeptides were not found to change the conformation when metal salts or ammonium salts such as Cl^?H₃N^?(CH₂)₁₁ COOEt, Gly-OEt-HCl, dl-Val-OEt-HCl and l-Leu-OEt-HCl were added, but a significant conformation change occurred upon adding d-Leu-OEt·HCl. If the same situation holds with the addition of α-amino acid p-nitrophenyl ester hydrochlorides, the previously reported enantiomer-selective catalysis by the tripeptides which hydrolysed d-Leu-OPh(NO₂·HCl faster than l-Leu-OPh(NO₂)·HCl can be explained; that is, the tripeptides change the conformation only when d-Leu-OPh(NO₂)·HCl is bound and consequently the intramolecular reaction is facilitated. This phenomenon may be compared with that of ‘induced fit’ in enzyme catalysis.     

Histidine-containing cyclic dipeptides as catalysts in the hydrolysis of carbonic acid p-nitrophenyl esters

A histidine-containing cyclic dipeptide, cyclo(D -Leu-L -His), was almost 20 times as efficient a catalyst as imidazole in the hydrolysis of p-nitrophenyl laurate. The effect of dioxane on the hydrolysis showed that hydrophobic interaction between the cyclic dipeptide and the ester is very important. This reaction obeyed the Michaelis-Menten kinetics, and the Michaelis constant K_m was as low as 9.98 × 10^?5M. Since the linear dipeptide having D -Leu-L -His sequence was nearly inactive in the hydrolysis, the functional groups of cyclo(D -Leu-L -His) in a specific arrangement held by the rigid backbone must have cooperated in the fast hydrolysis. Very weak catalysis by the diasteremeric cyclic dipeptide, cyclo(L -Leu-L -His), in the hydrolysis supported the above view.     

Solution conformation and hydrolytic activity of cyclo(D-leu-L-His)

It has been reported previously that a cyclic dipeptide, cyclo(D -Leu-L -His), showed a high hydrolytic activity toward a hydrophobic ester, p-nitrophenyl laurate. In order to determine the reason for the high catalytic activity, the conformation of cyclo(D -Leu-L -His) in aqueous solution was investigated by nuclear magnetic resonance and circular dichroism spectroscopy and compared with the conformation of cyclo(L -Leu-L -His), which was nearly inactive in otherwise the same conditions for the hydrolysis. It was demonstrated that the spatial arrangement of the hydrophobic isobutyl group of the D -leucyl residue and of the nucleophilic imidazolyl group of the L -histidyl residue in cyclo(D -Leu-L -His) matches very well with the long acyl chain and the active ester function of p-nitrophenyl laurate. On the other hand, in cyclo(L -Leu-L -His) the hydrophobic and the nucleophilic pendant groups are too close with each other to cooperate intramolecularly for the hydrolysis. It was concluded that the different steric structures of the diastereomers can explain the large difference of the catalytic activities.     

Solvolysis of charged active esters of carboxylic acids with cyclo (l- or d-Glu-l-His)

Cyclic dipeptide cyclo(l- or d-Glu-l-His) carrying an anionic site and a nucleophilic site has been synthesized and used as a catalyst for the solvolysis of cationic esters in aqueous alcohols. In the solvolysis of 3-acyloxy-N-trimethylanilinium iodide (S⁺_n, n = 2 and 10) and Cl^?H₃N⁺(CH₂)₁₁COOPh(NO₂), no efficient nucleophilic catalysis was observed. On the other hand, in the solvolysis of Gly-OPh(NO₂)·HCl, Val-OPh(NO₂)·HCl and Leu-OPh(NO₂)·HCl a very efficient general base-type catalysis by cyclo(l-Glu-l-His) was observed. In particular, with the latter two substrates the catalysis by cyclo(l-Glul-His) was more efficient than that by imidazole, although the catalysis was not enantiomer-selective. The diastereomeric cyclic dipeptide cyclo(d-Glu-l-His) was almost inactive under the same conditions. Confomation of cyclo(l- or d-Glu-l-His) in aqueous solution was investigated and the structure/catalysis relationship is discussed.     

Alpha-chymotrypsin: effects of bicyclic substrate geometry and hydrophobicity on reactivity

The α-chymotrypsin-catalyzed hydrolysis rates of p-nitrophenyl cyclopentane-carboxylate (I), p-nitrophenyl indan-2-carboxylate (II), and p-nitrophenyl spiro-[4.4]nonane-2-carboxylate (III) were measured at pH 8.1 in 20% methanol. After correction for variations in reactivity owing to stereoelectronic effects inherent to the substrates, the deacylation rate constants (k_c)_n of I and II are not significantly different. In $\text{(}\text{k}{}_{\mathrm{c}}\text{K}{}_{\mathrm{m}}\text{)}{}_{\mathrm{n}}$ II is 50 times more reactive than I, which demonstrates that the aromatic ring of the former substrate contributes significantly to its reactivity. The nearly equal reactivities of II and III indicate that the enzyme is rather insensitive to the geometry of the nonester-bearing ring of these compounds.     

Zinc enzyme modelling with zinc complexes of polar pyrazolylborate ligands

The Zn-OH₂ and Zn-OH complexes of the new tris(pyrazolyl)borate ligands with pyridyl and carboxamido substituents were investigated for their reactivity towards hydrolyzeable substrates. Tp^4−Py,MeZn-OH inserted CO₂ and CS₂ in methanol forming the Zn-OCOOMe and Zn-SCSOMe products. In non-aqueous media, both types of complexes with both types of substituents on the Tp ligands effected stoichiometric cleavage of tris(p-nitrophenyl)phosphate and p-nitrophenyl acetate. In solutions containing water and the MOPS buffer, up to eight p-nitrophenyl groups per equivalent of zinc complex could be cleaved from the esters, and the resulting bis(p-nitrophenyl)phosphate was also degraded to mono(p-nitrophenyl)phosphate. This is the first time that pyrazolylborate-zinc complexes have shown catalytic activity in hydrolytic reactions.     

Mechanistic role of each metal ion in Streptomyces dinuclear aminopeptidase: Peptide hydrolysis and 7 × 10-fold rate enhancement of phosphodiester hydrolysis

The dinuclear aminopeptidase from Streptomyces griseus (SgAP) and its metal derivatives catalyze the hydrolysis of the phosphoester bis(p-nitrophenyl) phosphate (BNPP) and the phosphonate ester p-nitrophenyl phenylphosphonate with extraordinary rate enhancements at pH 7.0 and 25 °C [A. Ercan, H. I. Park, L.-J. Ming, Biochemistry 45, (2006) 13779-13793.], reaching 6.7 billion-fold in terms of the first-order rate constant of the di-Co(II) derivative with respect to the autohydrolytic rates. Since phosphoesters are transition state-like inhibitors in peptide hydrolysis, their hydrolysis by SgAP is quite novel. Herein, we report the investigation of this proficient alternative catalysis of SgAP and the role of each metal ion in the dinuclear site toward peptide and BNPP hydrolysis. Mn(II) selectively binds to one of the dinuclear metal sites (M1), affording MnE-SgAP with an empty (E) second site for the binding of another metal (M2), including Mn(II), Co(II), Ni(II), Zn(II), and Cd(II). Peptide hydrolysis is controlled by M2, wherein the k_cat values for the derivatives MnM2-SgAP are different yet similar between MnCo- and CoCo-SgAP and pairs of other metal derivatives. On the other hand, BNPP hydrolysis is affected by metals in both sites. Thus, the two hydrolytic catalyses must follow different mechanisms. Based on crystal structures, docking, and the results presented herein, the M1 site is close to the hydrophobic specific site and the M2 site is next to Tyr246 that is H-bonded to a coordinated nucleophilic water molecule in peptide hydrolysis; whereas a coordinated water molecule on M1 becomes available as the nucleophile in phosphodiester hydrolysis.     

1H nuclear magnetic resonance studies of histidine-containing di- and tripeptides. Estimation of the effects of charged groups on the pKa value of the imidazole ring.

The nmr titration curves of chemical shifts versus pH were observed for the protons of various histidine-containing di- and tripeptides. With these results, the macroscopic pK_a values and the chemical shifts intrinsic to each ionic species were determined by a computer curve-fitting based on a simple acid dissociation sequence. The pK_a value of the imidazole ring in N-acetyl-L -histidine methylamide was assumed to represent the intrinsic (or unperturbed) pK_a of the imidazole rings of histidine having peptide linkages at both the CO and NH sides. The pK_a values of the imidazole rings observed for most di- and tripeptides were reasonably reproduced by simple calculations using the intrinsic value and the perturbations due to the CO₂^? and NH₃⁺ groups located at various positions. Some other factors affecting the pK_a value of the imidazole ring are also discussed.     

Hydroxo-complexes of Hg2+ chelates as models of hydrolytic metalloenzymes

Hydroxo-complexes of chelates of Hg²⁺ were found to catalyze hydrolysis of active esters and amides. Thus, 10⁻²M of the hydroxo-complex of Hg²⁺ pentamethyl diethylenetriamine (Hg-PDETA) at pH 7 enhanced the rate of hydrolysis of p-nitrophenyl esters and of cinnamoyl imidazole by factors of 400 and 1900, respectively. In the latter case the rates of reaction were linear with catalyst concentration. The hydroxo-complexes of Hg²⁺ phenanthroline (Hg-Phen) exhibited kinetic specificity toward p-nitrophenyl carbalkoxyglycinates. With these specific substrates acceleration factors of 1000 and more were obtained at 5 × 10⁻³M Hg-Phen, pH 8. The dependence of rates upon catalyst concentration was found to be curvilinear. This latter behavior was attributed to attack of one molecule of Hg-Phen, in the form of a hydroxo-complex, on a ternary complex Hg-Phen-substrate. The general features of metal-ion-catalyzed hydrolytic reactions are discussed and compared with the mode of action of hydrolytic metalloenzymes such as carboxypeptidase A and carbonic anhydrase.     

Specific spectrophotometric assays for cathepsin B1.

Cathepsin B₁ from bovine spleen was partially purified by acetone fractionation and by chromatography on Sephadex G-150 and DEAE Sephadex A-50. The enzyme was shown to catalyze the hydrolysis of p-nitrophenyl benzyloxycarbonylglycinate and p-nitrophenyl α-N-benzyloxycarbonyl-l-lysinate. Under the assay conditions, cathepsin B₁ is the major enzyme present in bovine spleen homogenates hydrolyzing these substrates. The kinetic parameters for the hydrolysis of p-nitrophenyl benzyloxycarbonylglycinate and p-nitrophenyl α-N-benzyloxycarbonyl-l-lysinate were measured and compared with those obtained for other cathepsin B₁ substrates. These results form the basis of an improved spectrophotometric assay for this enzyme in which the liberation of p-nitrophenol from either the N-benzyloxycarbonyl glycine or lysine p-nitrophenyl ester is monitored continuously at 326 nm.     

Transglycosylating and hydrolytic activities of the β-mannosidase from Trichoderma reesei

A purified β-mannosidase (EC 3.2.1.25) from the fungus Trichoderma reesei has been identified as a member of glycoside hydrolase family 2 through mass spectrometry analysis of tryptic peptides. In addition to hydrolysis, the enzyme catalyzes substrate transglycosylation with p-nitrophenyl β-mannopyranoside. Structures of the major and minor products of this reaction were identified by NMR analysis as p-nitrophenyl mannobiosides and p-nitrophenyl mannotriosides containing β-(1 → 4) and β-(1 → 3) linkages. The rate of donor substrate hydrolysis increased in presence of acetonitrile and dimethylformamide, while transglycosylation was weakly suppressed by these organic solvents. Differential ultraviolet spectra of the protein indicate that a rearrangement of the hydrophobic environment of the active site following the addition of the organic solvents may be responsible for this hydrolytic activation.     

The secretory nature of the excretory gland cells of Stephanurus dentatus. II. Presence of hydrolytic enzymes

Enzymes catalyzing the hydrolysis of casein, N-benzoyl-l-tyrosine ethyl ester, p-nitrophenyl acetate, and l-leucyl-β-naphthylamine hydrochloride were found in extracts of the excretory gland cells of Stephanurus dentatus.     

Ligand Binding to the FA3-FA4 Cleft Inhibits the Esterase-Like Activity of Human Serum Albumin

The hydrolysis of 4-nitrophenyl esters of hexanoate (NphOHe) and decanoate (NphODe) by human serum albumin (HSA) at Tyr411, located at the FA3-FA4 site, has been investigated between pH 5.8 and 9.5, at 22.0°C. Values of K _s, k ₊₂, and k ₊₂/K _s obtained at [HSA] ≥ 5×[NphOXx] and [NphOXx] ≥ 5×[HSA] (Xx is NphOHe or NphODe) match very well each other; moreover, the deacylation step turns out to be the rate limiting step in catalysis (i.e., k ₊₃ << k ₊₂). The pH dependence of the kinetic parameters for the hydrolysis of NphOHe and NphODe can be described by the acidic pK _a-shift of a single amino acid residue, which varies from 8.9 in the free HSA to 7.6 and 7.0 in the HSA:NphOHe and HSA:NphODe complex, respectively; the pK>_a-shift appears to be correlated to the length of the fatty acid tail of the substrate. The inhibition of the HSA-Tyr411-catalyzed hydrolysis of NphOHe, NphODe, and 4-nitrophenyl myristate (NphOMy) by five inhibitors (i.e., diazepam, diflunisal, ibuprofen, 3-indoxyl-sulfate, and propofol) has been investigated at pH 7.5 and 22.0°C, resulting competitive. The affinity of diazepam, diflunisal, ibuprofen, 3-indoxyl-sulfate, and propofol for HSA reflects the selectivity of the FA3-FA4 cleft. Under conditions where Tyr411 is not acylated, the molar fraction of diazepam, diflunisal, ibuprofen, and 3-indoxyl-sulfate bound to HSA is higher than 0.9 whereas the molar fraction of propofol bound to HSA is ca. 0.5.     

Multiple sugar binding sites in α-glucosidase

Twenty-five analogs of d-glucose were examined as reversible inhibitors of yeast α-glucosidase (EC 3.2.1.20). The K_i values range from 0.38 mM for 6-deoxy-d-glucose (quinovose) to 1.0 M for d-lyxose at pH=6.3 (0.1 M NaCl, 25°). All the monosaccharides and the three disaccharides (maltose, isomaltose and α,α-trehalose) were found to be linear competitive inhibitors with respect to α-p-nitrophenyl glucoside (pNPG) hydrolysis. Multiple inhibition analysis reveals that there are at least three monosaccharide binding sites on the enzyme. One of these can be occupied by glucose [K_i=1.8(±0.1) mM], one by d-galactose [K_i=164(±11) mM] and one by d-mannose [K_i=120(±9) mM]. The pH dependence for glucose binding closely follows that of V/K [pK_a1=5.55(±0.15), pK_a2=6.79(±0.15)], but the binding of mannose does not. Although the glucose subsite can be occupied simultaneously with the mannose or galactose subsites in the enzyme–product complex, no transglucosylation can be detected between pNPG and either mannose or galactose. This suggests that neither of these nonglucose subsites can be occupied in a productive manner in the covalent glucosyl-enzyme intermediate.     

On the physical properties and mechanism of action of arylsulfate sulfohydrolase II from Aspergillus oryzae.

Sedimentation equilibrium studies on arylsulfate sulfohydrolase II (EC 3.1.6.1) from Aspergillus oryzae under nondissociating conditions have resulted in a revised molecular weight of 94,900 ± 7100. Sedimentation equilibrium and gel electrophoresis data collected in the presence of the dissociating agents, urea and sodium dodecylsulfate demonstrate that the native enzyme is composed of two identical subunits as suggested by previous studies employing an irreversible inhibitor.The pH dependencies of the kinetic parameters V and $\text{V}\text{K}{}_{\mathrm{m}}$ for the enzymic hydrolysis of 4-nitrophenyl sulfate indicate that two groups of pK_a 4.7 and 6.0 control the activity of the enzyme. The product inorganic sulfate was shown to be a linear competitive inhibitor of the enzyme at pH 4.0, implying that it is a last released product along the reaction pathway. Inhibition by the phenol product was not observed. Enzymic hydrolysis of 4-nitrophenyl sulfate in ¹⁸O enriched water revealed that one atom of solvent oxygen is incorporated per molecule of inorganic sulfate, which is consistent with a mechanism featuring sulfur-oxygen bond cleavage. Evidence is presented based on stopped-flow kinetics, partitioning experiments in the presence of amine nucleophiles, and ¹⁸O exchange studies that collectively suggest that the breakdown of a covalent sulfuryl enzyme intermediate probably is not the rate-limiting step along the reaction pathway.The substrate specificity of the enzyme was examined by testing a variety of sulfate and phosphate esters as inhibitors of the hydrolysis of 4-nitrophenyl sulfate. The Cbz-l-Phe-l-Tyrosine-O-sulfate methyl ester serves as a substrate for the enzyme. Apparently substrate activity requires an aromatic sulfate ester whose binding is enhanced by incorporating the aromatic moiety in a hydrophobic matrix.     

Solvent isotope effects on α-glucosidase

The solvent kinetic isotope effects (SKIE) on the yeast α-glucosidase-catalyzed hydrolysis of p-nitrophenyl and methyl-d-glucopyranoside were measured at 25 °C. With p-nitrophenyl-d-glucopyranoside (pNPG), the dependence of k_cat/K_m on pH (pD) revealed an unusually large (for glycohydrolases) solvent isotope effect on the pL-independent second-order rate constant, ^DOD(k_cat/K_m), of 1.9 (±0.3). The two pK_as characterizing the pH profile were increased in D₂O. The shift in pK_a2 of 0.6 units is typical of acids of comparable acidity (pK_a=6.5), but the increase in pK_a1 (=5.7) of 0.1 unit in going from H₂O to D₂O is unusually small. The initial velocities show substrate inhibition (K_is/K_m～200) with a small solvent isotope effect on the inhibition constant [^DODK_is=1.1 (±0.2)]. The solvent equilibrium isotope effects on the K_is for the competitive inhibitors d-glucose and α-methyl d-glucoside are somewhat higher [^DODK_i=1.5 (±0.1)]. Methyl glucoside is much less reactive than pNPG, with k_cat 230 times lower and k_cat/K_m 5×10⁴ times lower. The solvent isotope effect on k_cat for this substrate [=1.11 (±0. 02)] is lower than that for pNPG [=1.67 (±0.07)], consistent with more extensive proton transfer in the transition state for the deglucosylation step than for the glucosylation step.     

Purification and characterization of novel cutinase from nasturtium (Tropaeolum majus) pollen.

Cutinase from pollen grains of Tropaeolum majus was purified by Sephadex G-100 gel filtration, QAE-Sephadex chromatography, and isoelectric focusing. The purified enzyme was homogeneous as judged by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate. The molecular weight of the enzyme was estimated to be 40,000 by both Sephadex G-100 gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This cutinase was found to be a glycoprotein containing about 7% carbohydrate and the isoelectric point of this enzyme was 5.45. It catalyzed hydrolysis of p-nitrophenyl esters of C₂ to C₁₈ fatty acids with similar K_m and V. The purified cutinase showed an optimum pH of 6.8 with cutin as the substrate, whereas with p-nitrophenyl esters of fatty acids the optimum pH was 8.0. This enzyme did not show any metal ion requirement. Unlike the previously studied fungal cutinases, the present pollen enzyme was strongly inhibited by thiol-directed reagents such as N-ethylmaleimide and p-hydroxymercuribenzoate whereas it was totally insensitive to the active serine-directed reagent, diisopropylfluorophosphate. The purified pollen cutinase showed preference for primary alcohol esters, but it did not catalyze hydrolysis of tripalmitoyl or trioleyl glycerol at significant rates. The properties of the pollen enzyme are, in general, in sharp contrast to those of the fungal cutinase, and the present results strongly suggest that the pollen enzyme belongs to a new class of cutinases. Another esterase which preferentially hydrolyzed p-nitrophenyl acetate was also found in the extracellular fluid. This enzyme, separated from cutinase, showed a pI of 5.6 and it was sensitive to diisopropylfluorophosphate, but not to SH-directed reagents.     

Human liver acid phosphatases: Purification and properties of a low-molecular-weight isoenzyme

A low-molecular-weight human liver acid phosphatase was purified 2580-fold to homogenity by a procedure involving ammonium sulfate fractionation, acid treatment, and SP-Sephadex ion-exchange chromatography with ion-affinity elution. The purified enzyme contains a single polypeptide chain and has a molecular weight of 14,400 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amino acid composition of this enzyme (E) is reported. A pH dependence study using p-nitrophenyl phosphate as a substrate (S) revealed the effect of substrate ionization (pK_a 5.2) and the participation of a group in the ES complex having a pK_a value of 7.8. The enzyme is readily inactivated by sulfhydryl reagents such as heavy metal ions. Alkylation of the enzyme with iodoacetic acid and iodoacetamide causes complete inactivation of the enzyme and this inactivation is prevented by the presence of phosphate ion. The enzyme is also inactivated by treatment with diethyl pyrocarbonate; protection against this reagent is afforded by phosphate ion. The substrate specificity of this enzyme is unusual for an acid phosphatase. Of the many alkyl and aryl phosphomonoesters tested, the only possibly physiological substrate hydrolyzed by this enzyme was flavin mononucleotide, which exhibits a V which is 3-fold larger at pH 5.0 and 6-fold larger at pH 7.0 than that for p-nitrophenyl phosphate. However, the enzyme also catalyzes the hydrolysis of acetyl phosphate at pH 5.0 with a velocity eight times larger than that reported for an acyl phosphatase from human erythrocytes.     

Esterolytic Properties of Leucine-Proteinase, the Leucine-Specific Serine Proteinase from Spinach (Spinacia oleracea L.) Leaves : A Steady-State and Pre-Steady-State Study

Steady-state and pre-steady-state kinetics for the hydrolysis of p-nitrophenyl esters of N-α-carbobenzoxy(-l-)amino acids catalyzed by leucine-proteinase were determined between pH 5 and 10 (I = 0.1 molar) at 23 ± 0.5°C. For the substrates considered: (a) the acylation step is rate-limiting in catalysis; (b) the pH profiles of k_cat and k_cat/K_m reflect the ionization of two groups with pK_a values ranging between 6.5 and 6.9, and 8.1 and 8.3 (probably, the histidine residue involved in the catalytic triad and the N-terminus, respectively); and (c) values of K_m are pH independent. Among the substrates examined, N-α-carbobenzoxy-l-leucine-p-nitrophenyl ester shows the most favorable catalytic parameters and allows to determine an enzyme concentration as low as 5 × 10⁻¹⁰ molar at the optimum pH value (approximately 7.5).     

Catalytic activity of linear-, cyclic- and polypeptides having a sequence of -Asp-β-Ala-Gly-ser-β-Ala-Gly-His-β-Ala-Gly in the hydrolysis of p-nitrophenyl acetate

Catalytic activities of Boc-Asp-β-Ala-Gly-Ser-β-Ala-Gly-His-β-Ala-Gly-OEt(Boc-9-Oet), Boc-Asp-β-Ala-Gly-Ser-β-Ala-Gly-His-β-Ala-Gly-OH(Boc-9-OH), cyclo(Asp-β-Ala-Gly-Ser-β-Ala-Gly-His-β-Ala-Gly) (Cyclic 9) and poly(Asp-β-Ala-Gly-Ser-β-Ala-Gly-His-β-Ala-Gly) (Poly 9) in the Hydrolysis of p-nitrophenyl acetate were investigated in detail and compared with each other and with poly(His-β-Ala-Gly) (Poly 3) which has no Ser and Asp residues. Generally, Poly 3 was less active than the others, which contain Ser and Asp residues together with the His residue. The reaction rate-substrate concentration for Boc-9-OEt, Boc-9-OH. Cyclic 9 and Poly 3 gave straight lines, while that for Poly 9 showed slightly the tendency of saturation at high substrate concentration. The reaction rates were all proportional to the concentration of the peptides. All peptides gave similar, sigmoid-type pH-k_cat profiles. The pK values obtained from these pH-k_cat profiles agreed fairly well with those of histidine residues obtained from ¹³C n.m.r. chemical shifts, which suggests that the predominant participating functional group in the catalytic reaction is the imidazole group in the histidine residue. The pK values of the His residue in peptides with the -Asp-β-Ala-Gly-Ser-β-Ala-Gly-His-β-Ala-Gly- sequence were shifted to higher pH region compared with Poly 3, suggesting that the effect of the carboxyl group in the Asp residue and the extents of pK-shift for linear peptides were larger than for Cyclic 9 or Poly 9. The catalytic reaction rates by Boc-9-OEt or Cyclic 9 increased steadily with increase in temperature, while the reaction rate-temperature profiles for Poly 9 and Poly 3 gave the optimum temperatures at around 40–50°C.