Kinetic study on chemical modification of Taka-amylase A. II. Ethoxycarbonylation of histidine residues

The modification of Taka-amylase A (TAA) [EC 3.2.1.1] of Aspergillus oryzae by diethylpyrocarbonate (DEP) was carried out at 25 degrees C and at pH 5.8 (0.1 M acetate buffer). Two out of the six histidine residues were modified with 4.6 mM DEP, and two or three histidine residues were modified with 23 mM DEP. In both cases, one of them was protected from modification by the presence of 15% maltose. The results suggest that two or three out of the six histidine residues are exposed on the surface of the TAA molecule, and one of them exists near the maltose binding site. Ethoxycarbonylation of histidine residues of TAA caused loss of the amylase activity and activation of the hydrolysis of phenyl alpha-maltoside (phi alpha M). The kinetic parameters of the modified TAA for several substrates and analogs were determined at 25 degrees C and at pH 5.3 (0.08 M acetate buffer). From the results, it was found that this alteration of the enzyme activity by the modification was not due to a change in Km value but to a change in k0 value. Thus, some of the histidine residues in TAA are suggested to play an important role in the enzyme catalytic function.     

Selective inhibition of histidine-modified pancreatic alpha-amylase by proteinaceous inhibitor from Phaseolus vulgaris

Chemical modification of two histidine residues of porcine pancreatic alpha-amylase (EC 3.2.1.1) by diethyl pyrocarbonate in the presence of a high concentration of maltotriose caused a decrease of amylase activity and an increase of maltosidase activity (hydrolysis of p-nitrophenyl-alpha-maltoside). By binding a proteinaceous inhibitor from Phaseolus vulgaris (white kidney bean) with the modified enzyme, the amylase activity was further decreased but the maltosidase activity was retained to about 100% that of the native enzyme. Both amylase and maltosidase activities of the native enzyme were almost completely inhibited by the proteinaceous inhibitor. The increase of maltosidase activity by histidine modification was due to an increase of kcat, whereas the Km value was not changed; but binding of the proteinous inhibitor affected mainly the Km value of the modified enzyme.     

Mechanism of interaction between milk xanthine oxidase and p-chloromercuribenzoate. Properties of the purified enzyme]

Two types of complexes are formed during the interaction of xanthine oxidase with p-chloromercurybenzoate (pCMB). The reversible inactive complex (presumably of absorption nature) is formed practically instantaneously and competitively with regard to the substrate (Ki=6,2 . 10(-8) M) in 0,05 M phosphate buffer (pH 7,8, 25 degrees) and does not involve the fast-reacting SH-groups of the enzyme. Reactivation of xanthine oxidase is observed during prolonged incubation of the inactive complex at 0 degrees; it is associated with the interaction between pCMB and the fact-reacting SH-groups. This interaction results in a dissociation of the inactive complex. The blocking of the slow-reacting SH-groups is accompanied by an irreversible loss of the xanthine oxidase activity. The enzyme modification by blocking of 10 fast-reacting SH-groups does not involve the Fe-S clusters, but results in local changes in the enzyme conformation. This is manifested in a 2-fold increase of Km and the rate constants of proteolysis of the modified xanthine oxidase as compared to the native enzyme. The rate constants of proteolysis by trypsin for the native and modified enzymes in 0,05 M phosphate buffer (pH 7,8; 37 degrees) are 3,7 . 10(-3) min-1 and 7,0 . 10(-3) min-1, respectively; those for chymotrypsin in the same buffer (30 degrees) are 1,5 . 10(-2) min-1 and 6,0 . 10(-2) min-1, respectively.     

Isosteric conversion of protein carboxyl groups into carboxamide groups. II. Application to lysozyme

For isosteric conversion of carboxyl groups of proteins into amide groups, ammonolysis of protein esters under mild conditions was attempted. Ammonolysis of methyl esters of lysozyme and bovine serum albumin proved to be incomplete. Highly reactive N-ethylsalicylamide esters of guanylated lysozyme were therefore prepared by subjecting the protein to reaction with N-ethylbenzisoxazolium ion at pH 4.2, 0 degree. Per molecule, 5-7 ester groups were introduced, with concomitant decrease of activity of 80-90%. Only 0.3 tyrosine was modified. On hydrolysis at pH 9.2 the activity was completely restored. At pH 7.9 three classes of ester groups could be distinguished: one group of high rate of hydrolysis (k1 = 1.5 min-1), three groups of intermediate rate (k2 = 0.13 min-1) and two groups of low rate (k3 = 0.018 min-1). The intermediate rate approximated the rate of hydrolysis of the model compound benzoylglycine N-ethylsalicylamide ester (k = 0.15 min-1). Ammonolysis at pH 9.2 in 2.0 M ammonia/ammonium acetate provided complete conversion of the ester groups into amide groups without restoration of activity, confirming the essentiality of certain carboxyl groups. In particular, rearrangement of the ester groups into relatively stable imide groups by O-N acyl migration was found to be completely absent. When native lysozyme was esterified with N-ethylbenzisoxazolium ion the activity did not completely return on hydrolysis.     

Substrate-selective activation of histidine-modified porcine pancreatic alpha-amylase by chloride ion.

Porcine pancreatic alpha-amylase (1,4-alpha-D-glucan glucanohydrolase) [EC 3.2.1.1] has both amylase activity (hydrolysis of alpha-1,4-D-glucoside bond of starch) and maltosidase activity (hydrolysis of p-nitrophenyl-alpha-D-maltoside to p-nitrophenol and maltose). By the modification of histidine residues of porcine pancreatic alpha-amylase with diethylpyrocarbonate (DEP), both amylase and maltosidase activities were decreased in the absence of chloride ion. In the presence of chloride ion, however, maltosidase activity of the modified enzyme was increased to more than 260% of that of the native enzyme, whereas amylase activity was decreased to less than 15% of the native enzyme. Since the chloride ion binding site is part of the active site loop [Buisson et al. (1987) Food Hydrocolloids 1,399-406 and Buisson et al. (1987) EMBO J. 6, 3909-3916], the special arrangements of both catalytic and modified histidine residues induced by the chloride ion binding would enhance only the maltosidase activity of the histidine-modified enzyme.     

A kinetic and fluorimetric investigation of papain modified at tryptophan-69 and -177 by N-bromosuccinimide

A systematic study of the modification of papain (its thiol group protected as a disulphide with mercaptoethanol) by N-bromosuccinimide, showed that 2 molar equiv. modified tryptophan-69 and 4 molar equiv. modified tryptophan-69 and -177. The Michaelis parameters for the catalysed hydrolysis of N-benzyloxycarbonylglycine p-nitrophenyl ester by these modified enzymes were determined. The enzymic activity of the modified enzymes was not seriously impaired, but modification of tryptophan-177 raised the apparent pK(a) of the acidic limb of the pH profile by more than 1 pH unit for both k(cat.) and k(cat.)/K(m). The fluorescence spectra (excitation at 288nm) of the modified enzymes showed that tryptophan-69 contributed about 8% to the fluorescence intensity, whereas tryptophan-177 contributed about 46% at neutral pH. However, the contribution of tryptophan-177 was quenched at low pH and its fluorescence intensity showed sigmoidal pH-dependence, with an apparent pK(a) of 4.2. Histidine-159, which is in close contact with tryptophan-177, is considered to be the residue responsible for the fluorescence quenching. When tryptophan-177 was modified, presumably generating a less hydrophobic micro-environment, the apparent pK(a) determined kinetically was raised to about 5.4. By comparing the Michaelis parameters of native papain, papain modified at tryptophan-69 and papain modified at tryptophan-69 and -177 with N-benzyloxycarbonylglycylglycine amide and N-benzyloxycarbonylglycyltryptophan amide, tryptophan-69 and tryptophan-177 were shown to be structural features of the S(2) and S(1)' subsites respectively.     

Chemical modification of Escherichia coli RNA polymerase by diethyl pyrocarbonate: evidence of histidine requirement for enzyme activity and intrinsic zinc binding

RNA polymerase (RPase) from Escherichia coli contains five subunits (alpha 2 beta beta' sigma) and two intrinsic Zn ions located in the beta and beta' subunits. This enzyme was rapidly inactivated by diethyl pyrocarbonate (DEP) at pH 6.0 and 25 degrees C. The difference spectrum of the DEP-inactivated and native RPases showed a single peak at 240 nm indicating the formation of N-carbethoxyhistidines. No decrease in absorbance at 278 nm, due to O-carbethoxytyrosine, or modification of amino and sulfhydryl groups was observed. Inactivated RPase with six to nine histidines being modified could be fully reactivated by incubation with 0.5 M hydroxylamine at pH 6.0 and room temperature for 1 h. No structural difference was detected between the native and modified enzymes as evidenced by UV/visible and fluorescence spectra, sodium dodecyl sulfate-polyacrylamide gel electrophoretic pattern, or gel filtration properties. Substrate ATP at 0.11 and 1.14 mM concentrations provided, respectively, 25% and 90% protection against DEP inactivation, while template DNA did not. These results suggest that one or more histidine residues is/are in close proximity to the substrate binding site. The pH dependence of the DEP inactivation of RPase suggested the modification of histidine at the active site with a pK value of 6.9. The inactivation of RPase by DEP and the formation of N-carbethoxyhistidine displayed a similar second-order rate constant of approximately 0.9 mM-1 min-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thiol groups of Escherichia coli citrate synthase and their influence on activity and regulation.

The modification of Escherichia coli citrate synthase (citrate oxaloacetatelyase(pro-3S-CH2.COO- leads to acetyl-CoA, EC 4.1.3.7) with 5,5'-dithiobis-(2-nitrobenzoic acid) has been investigated. (1) In low ionic strength (20 mM Tris.HCl, pH 8.0): (A) Eight thiol groups per tetramer of the native enzyme reacted with Nbs2. (b) Two of the eight accessible thiols were modified rapidly with the loss of 26% enzyme activity but with no change in the NADH inhibition. The remaining six were modified more slowly, resulting in a further 60% loss of activity and complete densensitization to NADH. (c) The 2nd-order rate constant for the modification of the rapidly reacting thiols is 2.5.10(4) M-1.min-1. At the reagent concentrations used (0.1 to 0.2 mM) the modification of the six thiols in the slow kinetic set appeared to be 1st-order; at 0.1 mM dithionitrobenzoic acid their rate of modification was approximately 30 times slower than the thiols in the fast kinetic set. (2) In high ionic strength (20 mM Tris.HCl, pH 8.0, 0.1 M KCl): (a) Four thiol groups were modified in a single kinetic set and it appeared that these thiols are four of the six slowly modified in the absence of KCl. (b) The modification resulted in 70% loss of enzyme activity and complete loss of NADH inhibition. (3) From the kinetic analysis it is proposed that the four thiol groups accessible to dithionitrobenzoic acid in the absence and presence of 0.1 M KCl are those involved in the response of NADH. Modification of any one of these four groups produced no reduction in the inhibition; instead, loss of NADH sensitivity was coincident with the appearance of tetrameric protein possessing three substituted thiols, whereas enzyme with one or two modified groups was still fully inhibited by NADH.     

The reaction of human alpha 2-macroglobulin with alpha-chymotrypsin. A stopped-flow kinetic investigation

The kinetics of the conformational changes of human alpha 2-macroglobulin (alpha 2M) induced by reaction with pure alpha-chymotrypsin, have been analyzed using three fluorescent probes, namely protein tryptophan groups and the dye 6-(4-toluidino)-2-naphthalenesulfonate, to monitor alterations of the alpha 2M structure, and a covalent conjugate of chymotrypsin and fluorescein isothiocyanate (Chy-FITC). The main reaction sequence exhibits a triphasic time course with any of the labels used. Each phase is first-order. The fixation of a single molecule of chymotrypsin to one protease-binding site of alpha 2M (site A) initiates the whole process and determines the access to the second site (site B). Of the three exponential phases of the reaction (20 degrees C), phase I (k1 approximately 19.6 min-1) and phase II (k2 approximately 5.3 min-1) belong to site A. Phase III is related to site B transformation. It contains two steps with different responses from tryptophan (k3 approximately 0.77 min-1) and Chy-FITC (k3 approximately 0.19 min-1) fluorescence measurements. The point to be stressed is that site A and site B, while presumably identical in the native form, are not equivalent with regard to their fluorescence and kinetic properties. However, the activation energy (E = 30.1 +/- 2.7 kJ mol-1) is the same for the three phases of the reaction. When present in sufficient excess, free chymotrypsin or native alpha 2M is able to form reversible complexes with the above-related chymotrypsin-alpha 2M adducts. Only the alpha 2M site A core seems to be involved in this parallel process. In addition the conformational state of the chymotrypsin-alpha 2M complexes is shown to depend on the pH, with a pKa of 6.4.     

3,4,5,6-Tetrahydrophthalic anhydride modification of glutamate dehydrogenase: the construction and activity of heterohexamers

Modification of glutamate dehydrogenase with 3,4,5,6-tetrahydrophthalic anhydride at pH 8.0 results in the progressive loss of enzymatic activity and a concomitant increase in the negative charge of the protein. Although the rate of inactivation at room temperature is too rapid to allow accurate rate constant determination, modification at 4 degrees C shows that the pseudo-first-order rate constant for inactivation appears to show a saturation effect with increasing reagent concentration, with a maximum of approximately 1 min-1. Control experiments showed that tetrahydrophthalic anhydride was hydrolyzed at a much slower rate, with a pseudo-first-order rate constant of 0.041 min-1. Protection studies indicated that inactivation was decreased by the active site ligands, NADP and 2-oxoglutarate. The extents of inactivation, whether assayed with glutamate at pH 7.0 or norvaline at pH 8.0, were the same. Changes in mobility on native gels and isoelectric point were used to follow the incorporated negative charge resulting from modification. Enzyme modified in the presence of protecting ligands (where activity is maintained) showed mobility changes which suggested that a single site of modification was protected. Modified enzyme incorporated 0.78 mol pyridoxal 5-phosphate less than native enzyme, consistent with modification of lysine-126. Enzyme modified under limiting conditions was shown to have a quaternary structure similar to that of the native enzyme, as judged by crosslinking patterns obtained with dimethylpimelimidate. The modified protein is readily resolved from unmodified protein using an NaCl double gradient elution from DEAE-Sephacel. The modification is reversed with regain of activity by incubation of the modified enzyme at low pH. We have made use of the recently demonstrated ability of guanidine hydrochloride to dissociate the hexamer of glutamate dehydrogenase into trimers that can then be reassociated to construct heterohexamers of glutamate dehydrogenase, in which one trimer of the heterohexamer contains native subunits while the other has been inactivated by the 3,4,5,6-tetrahydrophthalic anhydride modification. The heterohexamer is separated from either native or fully modified hexamers by DEAE-Sephacel chromatography. Significantly, the heterohexamer has little detectable catalytic activity, although activity is regained by reversal of the modification of the one modified trimer in the hexamer. This demonstrates that catalytic site cooperation between trimers in the hexamer of glutamate dehydrogenase is an essential component of the enzymatic activity of this enzyme.     

Chemical modification of lysine residues in Bacillus licheniformis alpha-amylase: conversion of an endo- to an exo-type enzyme

The lysine residues of Bacillus licheniformis alpha-amylase (BLA) were chemically modified using citraconic anhydride or succinic anhydride. Modification caused fundamental changes in the enzymes specificity, as indicated by a dramatic increase in maltosidase and a reduction in amylase activity. These changes in substrate specificity were found to coincide with a change in the cleavage pattern of the substrates and with a conversion of the native endo- form of the enzyme to a modified exo- form. Progressive increases in the productions of rho-nitrophenol or glucose, when para nitrophenyl-maltoheptaoside or soluble starch, respectively, was used as substrate, were observed upon modification. The described changes were affected by the size of incorporated modified reagent: citraconic anhydride was more effective than succinic anhydride. Reasons for the observed changes are discussed and reasons for the effectivenesses of chemical modifications for tailoring enzyme specificities are suggested.     

Kinetic study on chemical modification of taka-amylase A. I. Location and role of tryptophan residues

1. Five and four tryptophan residues in Taka-amylase A [EC 3.2.1.1] of A. oryzae (TAA) were modified with dimethyl(2-hydroxy-5-nitrobenzyl)-sulfonium bromide (K-IWS) in the absence and the presence of 15% maltose (substrate analog), respectively. Only one tryptophan residue was modified with dimethyl(2-methoxy-5-nitrobenzyl)-sulfonium bromide (K-IIWS) irrespective of the presence or absence of maltose. Kinetic parameters (molecular activity, k0, Michaelis constant, Km, and inhibitor constant, Ki) of the enzyme modified with K-IWS and K-IIWS were determined. The k0 value decreased with increase in the number of modified residues, but Km and Ki values and the type of inhibition were not altered by the modification. 2. The fluorescence quenching reaction of TAA with N-bromosuccinimide (NBS) proceeded in three phases. The second-order rate constants of the three phases were determined to be (4.3 +/- 0.5) x 10(5) M-1 . s-1, (2.1 +/- 0.3) x 10(3) M-1 . s-1 and (1.7 +/- 0.2) x 10(2) M-1 . s-1, respectively. In the presence of maltose, the first phase was further separated into two phases with rate constants of (4.6 +/- 0.6) x 10(6) M-1 . s-1 and (6.9 +/- 1.1) x 10(4) M-1 . s-1, respectively. On the basis of the results, it is estimated that five out of nine tryptophan residues are accessible to the solvent and among them, two tryptophan residues are substantially exposed: one is located in the maltose binding site near the catalytic site (its modification affects the catalytic function), and the other exists on the enzyme surface far from the active site.     

Inactivation of Pseudomonas iron-superoxide dismutase by hydrogen peroxide

Pseudomonas Fe-superoxide dismutase (superoxide:superoxide oxidoreductase, EC 1.15.1.1) is inactivated by hydrogen peroxide by a mechanism which exhibits saturation kinetics. The pseudo-first-order rate constant of the inactivation increased with increasing pH, with an inflection point around pH 8.5. Two parameters of the inactivation were measured in the pH range 7.8 to 9.0; the total H2O2 concentration at which the enzyme is half-saturated (K inact) was found to be independent of pH (30 mM) and the maximum rate constant for inactivation (k max) increased progressively with increasing pH, from 3.3 min-1 at pH 7.8 to 21 min-1 at pH 9.0. This evidence suggests the presence of an ionization group (pKa approximately 8.5) which does not participate in the binding of H2O2 but which affects the maximum inactivation rate of the enzyme. The loss of dismutase activity of the Fe-superoxide dismutase is accompanied by a modification of 1.6, 1.1 and 0.9 residues of tryptophan, histidine and cysteine, respectively. Since the amino acid residues of the Cr-substituted enzyme, which has no enzymatic activity, were not modified by H2O2, the active iron of the enzyme is essential for the modification of the amino acid residues.     

Phenoloxidase activity of intact and chemically modified functional unit RvH1: a from molluscan Rapana venosa hemocyanin

o-Diphenol oxidase activities (o-diPO) of chemically modified functional unit RvH1-a of molluscan hemocyanin Rapana venosa were studied using L-Dopa and dopamine as substrates. With L-Dopa as substrate the native FU RvH1-a did not show any o-diPO activity. Therefore the native FU RvH1-a was converted to enzymatic active form, after treatment with SDS, trypsin, urea and different values of pH when its o-diPO activity was studied. The highest artificial induction of o-diPO activity was observed after incubation of FU with 3.0mM SDS, and RvH1-a shows both, dopamine (K(M)=6.53mM, k(cat)/K(M)=1.29) and L-Dopa (K(M)=2.0mM, k(cat)/K(M)=2.1) activity due to a more open active site of the enzyme and better access of the substrates. It was determined that the K(M) value of SDS-activated RvH1-a against dopamine is higher compared to those of hemocyanins from Helix vulgaris, Helix pomatia and native tyrosinase from Ipomoea batatas but much lower than that from Illex argentinus (ST94) tyrosinase and arthropodan hemocyanin from Carcinus aestuarii. The Km value of SDS-activated RvH1-a against L-Dopa is higher than those of hemocyanins from H. vulgaris and Cancer magister, but lower than that of the tyrosinase from Streptomyces albus.     

Inhibition of enzymatic activity of alpha-thrombin by low molecular weight synthetic inhibitor]

The effect of the organophosphoric inhibitor, SA-152, on the fibrinogen-coagulating and TAME-esterase activity of bovine alpha-thrombin was studied. The irreversible inhibition constants (k11 = 1.1 x 10(4) M-1.min-1,Ki = 0.7 x 10(-4) M, k2 = 0.8 min-1 towards the coagulating activity and kII = 0.7 x 10(4) M-1.min-1, Ki = 0.3 x 10(-4) M, k2 = 0.2 min-1 towards the esterase activity) were determined. The SA-152 inactivated alpha-thrombin was dialyzed and incubated with 0.5 M and 2.5 M NaCl and 10 mM TAME. There was no reconstitution of activity of the SA-152 modified alpha-thrombin after dialysis and treatment with high concentrations of NaCl and TAME. Heparin interactions with the anion-binding site of the high molecular weight recognition center in the alpha-thrombin molecule did not significantly influence the values of the kinetic constants for the enzyme inhibition by SA-152. This finding is consistent with the hypothesis on the irreversible binding of SA-152 in the active center of the enzyme.     

Affinity labeling of the reduced diphosphopyridine nucleotide inhibitory site of glutamate dehydrogenase by 6-[(4-bromo-2,3-dioxobutyl)thio]-6-deaminoadenosine 5'-diphosphate

Bovine liver glutamate dehydrogenase reacts covalently with the new adenosine analogue 6-[(4-bromo-2,3-dioxobutyl)thio]-6-deaminoadenosine 5'-diphosphate with incorporation of about 1 mol of reagent/mol of enzyme subunit. Modified enzyme completely loses its normal ability to be inhibited by high concentrations of reduced diphosphopyridine nucleotide (DPNH) (greater than 100 microM), which binds at a regulatory site distinct from the catalytic site; however, the modified enzyme retains its full activity when assayed at 100 microM DPNH in the absence of allosteric compounds. The enzyme is still activated by ADP, is inhibited by GTP (albeit at higher concentrations), and binds 1.5-2 mol of [14C]GTP/subunit. A plot of initial velocity vs. DPNH concentration for the modified enzyme, in contrast to the native enzyme, followed Michaelis-Menten kinetics. The rate constant (k) for loss of DPNH inhibition (as measured at 0.6 mM DPNH) exhibits a nonlinear dependence on reagent concentration, suggesting a reversible binding of reagent (Kd = 0.19 mM) prior to irreversible modification. At 0.1 mM 6-[(4-bromo-2,3-dioxobutyl)thio]-6-deaminoadenosine 5'-diphosphate, k = 0.036 min-1 and is not affected by alpha-ketoglutarate, 100 microM DPNH, or GTP alone but is decreased to 0.0094 min-1 by 5 mM DPNH and essentially to zero by 5 mM DPNH plus 100 microM GTP. Incorporation after incubation with 0.25 mM 6-[(4-bromo-2,3-dioxobutyl)thio]-6-deaminoadenosine 5'-diphosphate for 2 h at pH 7.1 is 1.14 mol/mol of subunit in the absence but only 0.24 mol/mol of subunit in the presence of DPNH plus GTP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modification of histidines in human prothrombin. Effect on the interaction of fibrinogen with thrombin from diethyl pyrocarbonate-modified prothrombin

Diethyl pyrocarbonate (ethoxyformic anhydride) was used to modify histidyl residues in prothrombin. Diethyl pyrocarbonate inactivated the potential fibrinogen-clotting activity of prothrombin with a second-order rate constant of 70 M-1 min-1 at pH 6.0 and 25 degrees C. The difference spectrum of the modified protein had a maximum absorption at 240 nm which is characteristic of N-carbethoxyhistidine. The pH dependence for inactivation suggested the participation of a residue with a pKa of 6.2. Addition of hydroxylamine to ethoxyformylated prothrombin reversed the loss of fibrinogen-clotting activity. No structural differences were detected between the native and modified proteins using fluorescence emission and high-performance size-exclusion chromatography. The tyrosine and tryptophan content was not altered, but approximately 1-2 amino groups were modified. Statistical analysis of residual enzyme activity and extent of modification indicates that among 7 histidyl residues modified per molecule, there is 1 essential histidine (not in the active site) involved in the potential fibrinogen-clotting activity of prothrombin. To further examine its properties, the modified prothrombin was activated to thrombin using Echis carinatus venom protease. There was no difference in the catalytic activity of thrombin obtained from either native or ethoxyformylated prothrombin, as measured by H-D-Phe-pipecolyl-Arg-p-nitroanilide (D-Phe-Pip-Arg-NA) hydrolysis. However, thrombin produced from the modified protein showed a loss of fibrinogen-clotting activity but had a comparable apparent Ki value (about 20 microM) to thrombin from native prothrombin when fibrinogen was used as a competitive inhibitor during D-Phe-Pip-Arg-NA hydrolysis. The similarity in Ki values indicated that thrombin derived from diethyl pyrocarbonate-modified prothrombin does not have an altered fibrinogen-binding site. Although the histidyl residue involved during inactivation has not been identified, the results suggest that a histidyl residue in the thrombin portion of prothrombin is essential for interaction with fibrinogen.     

Identification of a histidyl residue in the active center of endoglucanase D from Clostridium thermocellum

Diethylpyrocarbonate modification of endoglucanase D from Clostridium thermocellum, cloned in Escherichia coli, resulted in a rapid but partial (maximally 70-80%) loss of activity. The second-order rate constant of inactivation proved to be exceptionally high (3210 M-1.min-1). A 3-fold reduction of the kcat and a 2-fold increase of the Km for 2'-chloro-4'-nitrophenyl beta-cellobioside were observed. Spectrophotometric analysis indicate the presence of one rapidly (k = 0.45 min-1) and two slower (k = 0.23 min-1) reacting histidyl residues. In the presence of 50 mM methyl beta-cellotrioside, the rate of inactivation was reduced 16-fold, and the kinetics of modification were compatible with the protection of 1 histidyl residue. Since peptide analysis was inconclusive, identification of the critical residue was attempted by site-directed mutagenesis. Each of the 12 histidyl residues present in the endoglucanase D sequence was mutated into either Ala or Ser. Seven of the mutant enzymes had specific activities lower than 50% of the wild-type. Only in the case of the Ser-516 mutant, however, was the residual activity not affected by diethyl pyrocarbonate. These findings suggest an important functional or structural role for His-516 in the wild-type enzyme.     

Improved pepsin inhibitor derived from activation peptide 1-16 of porcine pepsinogen.

The peptide Leu-Val-Lys-Val-Pro-Leu-Val-Arg-Lys-Lys-Ser-Leu-Arg-Gln-Asn-Leu, a known pepsin inhibitor, is derived from the first 16 amino acids of porcine pepsinogen. It was prepared from the activation mixture and was modified by guanidination of its three lysine residues to form homoarginine residues. The modified peptide is a better pepsin inhibitor than the native peptide; for 50% inhibition of the milk clotting action of pepsin at pH 5.3, the molar ratio of peptide to pepsin required is 9 for the native inhibitor and only 2 for the guanidinated inhibitor. The dissociation constants (k1) of the inhibitor-pepsin complexes are 7 X 10(-8) and 1.4 X 10(-8) M for the native and guanidinated peptides, respectively. The guanidinated peptide is more resistant to digestion by pepsin at pH 3.5. The native and modified peptides partially protect pepsin from inactivation at pH 7. Stepwise removal of the amino-terminal Leu-Val-Har residues from the guanidinated inhibitor by Edman degradation decreases the pepsin-inhibiting activity only slightly at the first step, but markedly at the second and third steps. Thus, all of the amino-terminal sequence except the leucine residue is necessary for full activity.