Amino acid residues involved in the catalytic mechanism of NAD-dependent glutamate dehydrogenase from Halobacterium salinarum

The pH dependence of kinetic parameters for a competitive inhibitor (glutarate) was determined in order to obtain information on the chemical mechanism for NAD-dependent glutamate dehydrogenase from Halobacterium salinarum. The maximum velocity is pH dependent, decreasing at low pHs giving a pK value of 7.19+/-0.13, while the V/K for l-glutamate at 30 degrees C decreases at low and high pHs, yielding pK values of 7.9+/-0.2 and 9.8+/-0.2, respectively. The glutarate pKis profile decreases at high pHs, yielding a pK of 9. 59+/-0.09 at 30 degrees C. The values of ionization heat calculated from the change in pK with temperature are: 1.19 x 10(4), 5.7 x 10(3), 7 x 10(3), 6.6 x 10(3) cal mol-1, for the residues involved. All these data suggest that the groups required for catalysis and/or binding are lysine, histidine and tyrosine. The enzyme shows a time-dependent loss in glutamate oxidation activity when incubated with diethyl pyrocarbonate (DEPC). Inactivation follows pseudo-first-order kinetics with a second-order rate constant of 53 M-1min-1. The pKa of the titratable group was pK1=6.6+/-0.6. Inactivation with ethyl acetimidate also shows pseudo-first-order kinetics as well as inactivation with TNM yielding second-order constants of 1.2 M-1min-1 and 2.8 M-1min-1, and pKas of 8.36 and 9.0, respectively. The proposed mechanism involves hydrogen binding of each of the two carboxylic groups to tyrosyl residues; histidine interacts with one of the N-hydrogens of the l-glutamate amino group. We also corroborate the presence of a conservative lysine that has a remarkable ability to coordinate a water molecule that would act as general base.     

Inactivation of Saccharomyces cerevisiae glucose-6-phosphate dehydrogenase by diethylpyrocarbonate

Glucose-6-phosphate dehydrogenase purified from Saccharomyces cerevisiae is rapidly inactivated by diethylpyrocarbonate at pH 6.8 and 30 degrees C with a concomitant increase in absorbance at 242 nm. The second-order rate constant for inactivation was calculated to be 487.8 M-1 min-1. The pH dependence of inactivation suggests the involvement of an amino acid residue having a pKa of 6.77. These results indicate that the inactivation is due to the modification of a histidine residue(s). In the presence of substrate, glucose-6-phosphate or NADP+, the rate of inactivation is decreased, indicating that the essential histidine residue(s) is located at the active site, possibly at the region of overlap of substrates at the binding site.     

Modification of uridine phosphorylase from Escherichia coli by diethyl pyrocarbonate. Evidence for a histidine residue in the active site of the enzyme.

Uridine phosphorylase from Escherichia coli is inactivated by diethyl pyrocarbonate at pH 7.1 and 10 degrees C with a second-order rate constant of 840 M-1.min-1. The rate of inactivation increases with pH, suggesting participation of an amino acid residue with pK 6.6. Hydroxylamine added to the inactivated enzyme restores the activity. Three histidine residues per enzyme subunit are modified by diethyl pyrocarbonate. Kinetic and statistical analyses of the residual enzymic activity, as well as the number of modified histidine residues, indicate that, among the three modifiable residues, only one is essential for enzyme activity. The reactivity of this histidine residue exceeded 10-fold the reactivity of the other two residues. Uridine, though at high concentration, protects the enzyme against inactivation and the very reactive histidine residue against modification. Thus it may be concluded that uridine phosphorylase contains only one histidine residue in each of its six subunits that is essential for enzyme activity.     

Identification of the essential histidine residue at the active site of Escherichia coli dehydroquinase.

The shikimate pathway enzyme 3-dehydroquinase is very susceptible to inactivation by the group-specific reagent diethyl pyrocarbonate (DEP). Inactivation follows pseudo first-order kinetics and exhibits a second-order rate constant of 148.5 M-1 min-1. An equilibrium mixture of substrate and product substantially protects against inactivation by DEP, suggesting that residues within the active site are being modified. Complete inactivation of the enzyme correlates with the modification of 6 histidine residues/subunit as determined by difference spectroscopy at 240 nm. Enzymic activity can be restored by hydroxylamine treatment, which is also consistent with the modification occurring at histidine residues. Using the kinetic method of Tsou (Tsou, C.-L. (1962) Sci. Sin. 11, 1535-1558), it was shown that modification of a single histidine residue leads to inactivation. Ligand protection experiments also indicated that 1 histidine residue was protected from DEP modification. pH studies show that the pKa for this inactivation is 6.18, which is identical to the single pKa determined from the pH/log Vmax profile for the enzyme. A single active site peptide was identified by differential peptide mapping in the presence and absence of ligand. This peptide was found to comprise residues 141-158; of the 2 histidines in this peptide (His-143 and His-146), only one, His-143, is conserved among all type I dehydroquinases. We propose that His-143 is the active site histidine responsible for DEP-mediated inactivation of dehydroquinase and is a good candidate for the general base that has been postulated to participate in the mechanism of this enzyme.     

High-resolution structure of the temperature-sensitive mutant of phage lysozyme, Arg 96----His

The structure of the temperature-sensitive mutant lysozyme of bacteriophage T4 in which arginine 96 is replaced by histidine has been determined crystallographically and refined to a residual of 17.6% at 1.9-A resolution. Overall, the three-dimensional structure of the mutant protein is extremely similar to that of wild type. There are local distortions in the mutant structure suggesting that the substituted His 96 residue is under strain. This appears to be one of the major reasons for the decreased thermostability. In wild-type lysozyme the guanidinium of Arg 96 is located at the carboxy terminus of alpha-helix 82-90 and makes a pair of hydrogen bonds to two of the carbonyl groups in the last turn of the helix. The loss of this "helix dipole" interaction also appears to contribute to the destabilization. The pKa* of His 96 in the mutant lysozyme has been determined by nuclear magnetic resonance and found to be 6.8 at 10 degrees C. This relatively normal value of the histidine pKa* suggests that the protonated and unprotonated forms of the imidazole ring are perturbed equally by the protein environment or, what is equivalent, the mutant lysozyme is equally stable with either histidine species.     

Inactivation of Acinetobacter calcoaceticus acetate kinase by diethylpyrocarbonate

Acetate kinase purified from Acinetobacter calcoaceticus was inhibited by diethylpyrocarbonate with a second-order rate constant of 620 M-1.min-1 at pH 7.4 at 30 degrees C and showed a concomitant increase in absorbance at 240 nm due to the formation of N-carbethoxyhistidyl derivative. Activity could be restored by hydroxylamine and the pH curve of inactivation indicates the involvement of a residue with a pKa of 6.64. Complete inactivation of acetate kinase required the modification of seven residues per molecule of enzyme. Statistical analysis showed that among the seven modifiable residues, only one is essential for activity. 5,5'-dithiobis(2-nitrobenzoic acid), p-chloromercuryphenylsulfonate, N-ethylmaleimide and phenylglyoxal did not affect the enzyme activity. These results suggest that the inactivation is due to the modification of one histidine residue. The substrates, acetate and ATP, protected the enzyme against inactivation, indicating that the modified histidine residue is located at or near the active site.     

Chemical modification of prostaglandin H synthase with diethyl pyrocarbonate.

The role of histidine in catalysis by prostaglandin H synthase has been investigated using chemical modification with diethyl pyrocarbonate (DEPC), an agent that has been found to rather selectively derivatize histidine residues in proteins under mild conditions. Incubation of the synthase apoprotein with DEPC at pH 7.2 resulted in a progressive loss of the capacity for both cyclooxygenase and peroxidase catalytic activities. The kinetics of inactivation of the cyclooxygenase activity were dependent on the concentration of DEPC; a second-order rate constant of 680 M-1 min-1 was estimated for reaction of the apoenzyme at pH 7.2 and 0 degrees C. The kinetics of inactivation of the cyclooxygenase by DEPC exhibited a sigmoidal dependence on the pH, indicating that deprotonation of a group with a pKa of 6.3 was required for inactivation. The presence of the heme prosthetic group slowed, but did not prevent, inactivation by DEPC. The stoichiometry of histidine modification of apoenzyme during inactivation determined from absorbance increases at 242 nm agreed well with the overall stoichiometry of derivatized residues determined with [14C]DEPC, indicating that modification by DEPC was quite selective for histidine residues on the synthase. Although modification of several histidine residues by DEPC was observed, only one of the histidine residues was essential for cyclooxygenase activity. Modification of the holoenzyme with DEPC altered the EPR signal of the hydroperoxide-induced tyrosyl free radical from the wide doublet (35 G, peak-to-trough) found with the native synthase to a narrower singlet (28 G, peak-to-trough) quite like that found in the indomethacin-synthase complex. Reaction of the indomethacin-synthase complex with DEPC was found to increase the cyclooxygenase velocity by 9 times its initial value, to about one-third of the uninhibited value, without displacement of the indomethacin; the peroxidase was significantly inactivated under the same conditions. Histidyl residues in the synthase are thus likely to have important roles not only in cyclooxygenase and peroxidase catalysis but also in the interaction of the synthase with indomethacin.     

Identification of amino acid residues essential for enzyme activity of sheep liver 5,10-methylenetetrahydrofolate reductase.

Sheep liver 5,10-methylenetetrahydrofolate reductase was subjected to specific chemical modification with phenylglyoxal, diethyl pyrocarbonate and N-bromosuccinimide. The second-order rate constants for inactivation were calculated to be 54 M-1 X min-1, 103 M-1 X min-1 and 154 M-1 X min-1 respectively. This inactivation could be prevented by incubation with substrates or products, suggesting that the residues modified, namely arginine, histidine and tryptophan, are essential for enzyme activity.     

Identification of serine and histidine adducts in complexes of trypsin and trypsinogen with peptide and nonpeptide boronic acid inhibitors by 1H NMR spectroscopy.

We have previously shown, in 15N NMR studies of the enzyme's active site histidine residue, that boronic acid inhibitors can form two distinct types of complexes with alpha-lytic protease. Inhibitors that are structural analogs of good alpha-lytic protease substrates form transition-state-like tetrahedral complexes with the active site serine whereas those that are not form complexes in which N epsilon 2 of the active site histidine is covalently bonded to the boron of the inhibitor. This study also demonstrated that the serine and histidine adduct complexes exhibit quite distinctive and characteristic low-field 1H NMR spectra [Bachovchin, W. W., Wong, W. Y. L., Farr-Jones, S., Shenvi, A. B., & Kettner, C. A. (1988) Biochemistry 27, 7689-7697]. Here we have used low-field 1H NMR diagnostically for a series of boronic acid inhibitor complexes of trypsin and trypsinogen. The results show that H-D-Val-Leu-boroArg and Ac-Gly-boroArg, analogs of good trypsin substrates, form transition-state-like serine adducts with trypsin, whereas the nonsubstrate analog inhibitors boric acid, methane boronic acid, butane boronic acid, and triethanolamine borate all form histidine adducts, thereby paralleling the previous results obtained with alpha-lytic protease. However, with trypsinogen, Ac-Gly-boroArg forms predominantly a histidine adduct while H-D-Val-Leu-boroArg forms both histidine and serine adducts, with the histidine adduct predominating below pH 8.0 and the serine adduct predominating above pH 8.0.(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.     

An active-site lysine in avian liver phosphoenolpyruvate carboxykinase

The participation of lysine in the catalysis by avian liver phosphoenolpyruvate carboxykinase was studied by chemical modification and by a characterization of the modified enzyme. The rate of inactivation by 2,4-pentanedione is pseudo-first-order and linearly dependent on reagent concentration with a second-order rate constant of 0.36 +/- 0.025 M-1 min-1. Inactivation by pyridoxal 5'-phosphate of the reversible reaction catalyzed by phosphoenolpyruvate carboxykinase follows bimolecular kinetics with a second-order rate constant of 7700 +/- 860 M-1 min-1. A second-order rate constant of inactivation for the irreversible reaction catalyzed by the enzyme is 1434 +/- 110 M-1 min-1. Treatment of the enzyme with pyridoxal 5'-phosphate gives incorporation of 1 mol of pyridoxal 5'-phosphate per mole of enzyme or one lysine residue modified concomitant with 100% loss in activity. A stoichiometry of 1:1 is observed when either the reversible or the irreversible reactions catalyzed by the enzyme are monitored. A study of kobs vs pH suggests this active-site lysine has a pKa of 8.1 and a pH-independent rate constant of inactivation of 47,700 M-1 min-1. The phosphate-containing substrates IDP, ITP, and phosphoenolpyruvate offer almost complete protection against inactivation by pyridoxal 5'-phosphate. Modified, inactive enzyme exhibits little change in Mn2+ binding as shown by EPR. Proton relaxation rate measurements suggest that pyridoxal 5'-phosphate modification alters binding of the phosphate-containing substrates. 31P NMR relaxation rate measurements show altered binding of the substrates in the ternary enzyme.Mn2+.substrate complex. Circular dichroism studies show little change in secondary structure of pyridoxal 5'-phosphate modified phosphoenolpyruvate carboxykinase. These results indicate that avian liver phosphoenolpyruvate carboxykinase has one reactive lysine at the active site and it is involved in the binding and activation of the phosphate-containing substrates.     

The reaction mechanism of the novel vanadium-bromoperoxidase. A steady-state kinetic analysis

The reaction of vanadium-bromoperoxidase from the brown alga Ascophyllum nodosum with hydrogen peroxide, bromide, and 2-chlorodimedone has been subjected to an extensive steady-state kinetic analysis. Systematic variation of pH and the concentrations of these three components demonstrate that the reaction model includes four enzyme species: native bromoperoxidase, a bromoperoxidase-bromide inhibitory complex, a bromoperoxidase-hydrogen peroxide intermediate, and a bromoperoxidase-HOBr species. This latter intermediate did not display any direct interaction with the nucleophilic reagent as oxidized bromine species (Br-3, Br2, and/or HOBr) were the primary reaction products. The generation of oxidized bromine species was as fast as the bromination of 2-chlorodimedone. The enzyme did not show any specificity with regard to bromination of various organic compounds. Formation of the bromoperoxidase-bromide inhibitory complex was competitive with the reaction between hydrogen peroxide and enzyme. From the steady-state kinetic data lower limits for the second-order rate constants at various pH values were calculated for individual steps in the catalytic cycle. This pH study showed that native enzyme must be unprotonated prior to binding of hydrogen peroxide (second-order association rate constant of 2.5.10(6) M-1.s-1 at pH greater than 6). The pKa for the functional group controlling the binding of hydrogen peroxide was 5.7 and is ascribed to a histidine residue. The reaction rate between bromide and enzyme-hydrogen peroxide intermediate also depended on pH (second-order association rate constant of 1.7.10(5) M-1.s-1 at pH 4.0).     

A chemical approach to the fine structure of biomolecular complexes: the amino terminal region of the 50S ribosomal "A" protein from Bacillus stearothermophilus.

An experimental approach and methodology are described for determining the reactive properties and ionization constants of individual functional groups of proteins within biomolecular complexes. The ionization constants and reactivities of the methionyl-l amino terminus and the lysyl-3 residue of the alanine rich 50S ribosomal "A" protein from Bacillus stearothermophilus have been determined by an extension of the competitive labeling technique used by H. Kaplan, K. J. Stevenson, and B. S. Hartley ((1971), Biochem. J. 124, 289-299). This approach employs (1-14C)- and (3H)acetic anhydride in a double-labeling procedure. In 0.1 M KCl-0.02 M Mg2+-0.05 M Veronal at 10 degrees the methionyl-l amino terminus has a pKa of 7.5 and is exposed on the surface of the ribosome. The lysyl-3 has a pKa of 10 and is also exposed to solvent at the surface of the 50S subunit. Based on a linear free energy relationship (Bronsted plot) obtained with a series of standard amines the methionyl amino terminus has a substantially higher reactivity than expected from its ionization constant. The lysyl epsilon-amino group has the expected reactivity. The abnormally high reactivity of the methionyl amino terminus can only be accounted for by a specific interaction with other functional groups in the ribosome. These data support the proposal that the charged state of this residue is important in the structure and function of the "A" protein at the surface of the ribosome.     

A steady-state study on the formation of Compounds II and III of myeloperoxidase

The reaction between native myeloperoxidase and hydrogen peroxide, yielding Compound II, was investigated using the stopped-flow technique. The pH dependence of the apparent second-order rate constant showed the existence of a protonatable group on the enzyme with a pKa of 4.9. This group is ascribed to the distal histidine imidazole, which must be deprotonated to enable the reaction of Compound I with hydrogen peroxidase to take place. The rate constant for the formation of Compound II by hydrogen peroxide was 3.5.10(4) M-1.s-1. During the reaction of myeloperoxidase with H2O2, rapid reduction of added cytochrome c was observed. This reduction was inhibitable by superoxide dismutase, and this demonstrates that superoxide anion radicals are generated. When potassium ferrocyanide was used as an electron donor to generate Compound II from Compound I, the pH dependence of the apparent second-order rate constant indicated involvement of a group with a pKa of 4.5. However, with ferrocyanide as an electron donor, protonation of the group was necessary to enable the reaction to take place. The rate constant for the generation of Compound II by ferrocyanide was 1.6.10(7) M-1.s-1. We also investigated the reaction of Compound II with hydrogen peroxide, yielding Compound III. Formation of Compound III (k = 50 M-1.s-1) proceeded via two different pathways, one of which was inhibitable by tetranitromethane. We further investigated the stability of Compound II and Compound III as a function of pH, ionic strength and enzyme concentration. The half-life values of both Compound II and Compound III were independent of the enzyme concentration and ionic strength. The half-life value of Compound III was pH-dependent, showing a decreasing stability with increasing pH, whereas the stability of Compound II was independent of pH over the range 3-11.     

Histidine decarboxylase of Lactobacillus 30a: function and reactivity of sulfhydryl groups.

Two classes of sulfhydryl groups in histidine decarboxylase from Lactobacillus 30 a can be differentiated by their reaction with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). Five cysteinyl residues (class I) of the native enzyme are titrated by DTNB as the pH of the reaction medium is increased from 6.5 to 7.5; the pH-rate profile for their reaction is described by a pKa of 9.2. An additional five thiol groups (class II) are titrated only when denaturing agents are added above neutral pH. Histidine decarboxylase is completely inactivated by DTNB in a kinetically second-order process (Kapp = 660 +/- 20 M-1 min-1 at pH 7.6 and 25 degrees C) which occurs coincident with and at the same rate as modification of the five class-I SH groups of the enzyme, i.e., one thiol group per pyruvoyl prosthetic group. The competitive inhibitors, histamine and imidazole, markedly enhanced the reactivity of these cysteinyl residues toward DTNB; this enhancement is accompanied by a concomitant increase in the rate of inactivation. A single SH group in each of the five catalytic units of histidine decarboxylase is thus implicated as being critical for the expression of enzymatic activity.     

Evidence for an essential histidine in neutral endopeptidase 24.11

Rat kidney neutral endopeptidase 24.11, "enkephalinase", was rapidly inactivated by diethyl pyrocarbonate under mildly acidic conditions. The pH dependence of inactivation revealed the modification of an essential residue with a pKa of 6.1. The reaction of the unprotonated group with diethyl pyrocarbonate exhibited a second-order rate constant of 11.6 M-1 s-1 and was accompanied by an increase in absorbance at 240 nm. Treatment of the inactivated enzyme with 50 mM hydroxylamine completely restored enzyme activity. These findings indicate histidine modification by diethyl pyrocarbonate. Comparison of the rate of inactivation with the increase in absorbance at 240 nm revealed a single histidine residue essential for catalysis. The presence of this histidine at the active site was indicated by (a) the protection of enzyme from inactivation provided by substrate and (b) the protection by the specific inhibitor phosphoramidon of one histidine residue from modification as determined spectrally. The dependence of the kinetic parameter Vmax/Km upon pH revealed two essential residues with pKa values of 5.9 and 7.3. It is proposed that the residue having a kinetic pKa of 5.9 is the histidine modified by diethyl pyrocarbonate and that this residue participates in general acid/base catalysis during substrate hydrolysis by neutral endopeptidase 24.11.     

The reaction of Aspergillus niger catalase with methyl hydroperoxide.

The formation of Compound I from Aspergillus niger catalase and methyl hydroperoxide (CH3OOH) has been investigated kinetically by means of rapid-scanning stopped-flow techniques. The spectral changes during the reaction showed distinct isobestic points. The second-order rate constant and the activation energy for the formation of Compound I were 6.4 x 10(3) M-1s-1 and 10.4 kcal.mol-1, respectively. After formation of Compound I, the absorbance at the Soret peak returned slowly to the level of ferric enzyme with a first-order rate constant of 1.7 x 10(-3) s-1. Spectrophotometric titration of the enzyme with CH3OOH indicates that 4 mol of peroxide react with 1 mol of enzyme to form 1 mol of Compound I. The amount of Compound I formed was proportional to the specific activity of the catalase. The irreversible inhibition of catalase by 3-amino-1,2,4-triazole (AT) was observed in the presence of CH3OOH or H2O2. The second-order rate constant of the catalase-AT formation in CH3OOH was 3.0 M-1 min-1 at 37 degrees C and pH 6.8 and the pKa value was estimated to be 6.10 from the pH profile of the rate constant of the AT-inhibition. These results indicate that A. niger catalase forms Compound I with the same properties as other catalases and peroxidases, but the velocity of the Compound I formation is lower than that of the others.     

Reaction of 5-enol-pyruvoylshikimate-3-phosphate synthase with diethyl pyrocarbonate: evidence for an essential histidine residue

5-enol-Pyruvoylshikimate-3-phosphate synthase catalyzes the reversible condensation of phosphoenolpyruvate and shikimate 3-phosphate to yield 5-enol-pyruvoylshikimate 3-phosphate and inorganic phosphate. The enzyme is a target for the nonselective herbicide glyphosate (N-phosphonomethylglycine). Diethyl pyrocarbonate inactivated this enzyme with a second-order rate constant of 220 M-1 min-1 at pH 7.0 and 0 degrees C. The rate of inactivation is pH dependent and the pH inactivation rate data show the involvement of a group with a pKa of 6.8. Almost all of the original activity was recovered by treatment of the inactivated enzyme with hydroxylamine. The difference spectrum of the inactivated and native enzyme reveals a single peak at 242 nm but no trough at around 278 nm is observed. Complete inactivation required the modification of four histidine residues per molecule of the enzyme. However, statistical analysis of the residual activity and the extent of modification shows that among the four modifiable residues, only one is critical for activity. Furthermore, this inactivation is prevented by the substrates of the enzyme. The above results indicated that one histidine is located within or very close to the active site and may play an important role in catalysis.     

Evidence for essential histidine and cysteine residues in calcium/calmodulin-sensitive cyclic nucleotide phosphodiesterase.

Ca/calmodulin-sensitive cyclic nucleotide phosphodiesterase (CaM-PDE) is an important enzyme regulating cGMP levels and relaxation of vascular smooth muscle. This modification study was conducted mostly with bovine brain CaM-PDE to identify essential functional groups involved in catalysis. The effect of pH on Vmax/Km indicates two essential residues with pKa values of 6.4 and 8.2. Diethyl pyrocarbonate (DEP), a histidine-modifying agent, inhibits CaM-PDE with a second-order rate constant of 130 M-1 min-1 at pH 7.0 and 30 degrees C. Activity is restored by NH2OH. The pH dependence of inactivation reveals that the essential residue modified by DEP has an apparent pKa of 6.5. The difference spectrum of the intact and DEP-treated enzyme shows a maximum between 230 and 240 nm, suggesting formation of carbethoxy derivatives of histidine. The enzyme is also inactivated by N-ethylmaleimide (NEM) and 5,5'-dithiobis-(2-nitrobenzoic acid), both sulfhydryl-modifying agents, with the latter effect reversed by dithiothreitol, which suggests inactivation resulting from modification of cysteine residue(s). Partial inactivation of the enzyme by DEP or NEM results in an apparent decrease in the Vmax without a change in the Km or the extent of CaM stimulation. The rate of inactivation by DEP is greater in the presence than in the absence of Ca/CaM. A substrate analogue, Br-cGMP, and the competitive inhibitor 3-isobutyl-1-methylxanthine partially protect the enzyme against inactivation by DEP or NEM, suggesting that the modification of histidine and cysteine residues occurs at or near the active site. DEP also inactivated porcine brain CaM-PDE.(ABSTRACT TRUNCATED AT 250 WORDS)     

The kinetics of hemostatic enzyme-antithrombin interactions in the presence of low molecular weight heparin

The kinetics of inhibition of four hemostatic system enzymes by antithrombin were examined as a function of heparin concentration. Plots of the initial velocity of factor Xa-antithrombin or plasmin-antithrombin interaction versus the level of added mucopolysaccharide exhibit an ascending limb and subsequent plateau regions. In each case, the kinetic profile is closely correlated with the concentration of the heparin . antithrombin complex formed within the reaction mixture. A decrease in the velocity of inhibition is not observed at high levels of added mucopolysaccharide despite the generation of significant quantities of heparin-enzyme interaction products. The second-order rate constants for the neutralization of factor Xa or plasmin by the mucopolysaccharide . inhibitor complex are 2.4 x 10(8) M-1 min-1 and 4.0 x 10(6) M-1 min-1, respectively. These parameters must be contrasted with the similarly designated constants obtained in the absence of heparin which are 1.88 x 10(5) M-1 min-1 and 4.0 x 10(4) M-1 min-1, respectively. Plots of the initial velocity of the factor IXa-antithrombin or the thrombin-antithrombin interaction versus the level of added mucopolysaccharide exhibit an ascending limb, pseudoplateau, descending limb, and final plateau regions. In each case, the ascending limb and pseudoplateau are closely correlated with the concentration of heparin c antithrombin complex formed within the reaction mixture. Furthermore, the descending limb and final plateau of these two processes coincide with the generation of increasing amounts of the respective mucopolysaccharide-enzyme interaction products. The second-order rate constants for the neutralization of factor IXa or thrombin by the heparin . antithrombin complex are 3.0 x 10(8) M-1 min-1 and 1.7 x 10(9) M-1 min-1, respectively. The second-order rate constants for the inhibition of mucopolysaccharide-factor IXa or mucopolysaccharide-thrombin interaction products by the heparin . antithrombin complex are 2.0 x 10(7) M-1 min-1 and 3.0 x 10(8) M-1 min-1, respectively. These kinetic parameters must be contrasted with similarly designated constants obtained in the absence of mucopolysaccharide which are 2.94 x 10(4) M-1 min-1 and 4.25 x 10(5) M-1 min-1, respectively. Thus, our data demonstrate that binding of heparin to antithrombin is required for the mucopolysaccharide-dependent enhancement in the rates of neutralization of thrombin, factor IXa, factor Xa, or plasmin by the protease inhibitor. Furthermore, a careful comparison of the various constants suggests that the direct interaction between heparin and antithrombin may be largely responsible for the kinetic effect of this mucopolysaccharide.