Phosphoprotein phosphatase activity of human prostate acid phosphatase

Human prostate acid phosphatase (EC 3.1.3.2) has been shown to dephosphorylate different phosphoproteins with the maximum rate at pH 4.0-4.5. The activity with phosvitin is distinctly higher than with beta-casein, casein and most of all than with riboflavin-binding protein. The native phosvitin is homogeneous on isoelectric focusing with pI value of 2.1, whereas phosvitin partially dephosphorylated (in about 15%) by the prostate acid phosphatase shows multiple bands with pI values of 3.5 - 6.8 or higher. The phosphate groups bound to serine residues are removed enzymatically twice as fast as phosphothreonine residues. The apparent Km value for phosvitin was 2.4 X 10(-7) M, and is by three orders of magnitude lower than Km of p-nitrophenyl phosphate (2.9 X 10(-4) M). The competitive inhibitors of prostate acid phosphatase, fluoride and L(+)-tartrate, show the same Ki values for phosvitin and p-nitrophenyl phosphate.     

Chemical replacement of P1' arginine residue at the first reactive site of peanut protease inhibitor B-III

The contribution of the P1' residue at the first reactive site of peanut protease inhibitor B-III to the inhibition was analyzed by replacement of the P1' Arg(11) with other amino acids (Arg, Ser, Ala, Leu, Phe, Asp) after selective modification of the second reactive site. The Arg derivative had the same trypsin inhibitory activity as the native inhibitor (Ki = 2 X 10(-9) M). The Ser derivative inhibited more weakly (Ki = 2 X 10(-8) M). The Ala and Leu derivatives inhibited trypsin very weakly (Ki = 2 X 10(-7) M and 4 X 10(-7) M, respectively), and the Phe and Asp derivatives not at all. These results suggest that the P1' arginine residue is best for inhibitory activity at the first reactive site of B-III, although it has been suggested that a P1' serine residue at the reactive site is best for inhibitory activity of Bowman-Birk type inhibitors.     

Purification and amino acid sequence of Kunitz-type protease inhibitor found in the hemocytes of horseshoe crab (Tachypleus tridentatus)

A low molecular weight protein protease inhibitor was purified from Japanese horseshoe crab (Tachypleus tridentatus) hemocytes. It consisted of a single polypeptide with a total of 61 amino acid residues. This protease inhibitor inhibited stoichiometrically the amidase activity of trypsin (Ki = 4.60 X 10(-10) M), and also had inhibitory effects on alpha-chymotrypsin (Ki = 5.54 X 10(-9) M), elastase (Ki = 7.20 X 10(-8) M), plasmin, and plasma kallikrein. However, it had no effect on T. tridentatus clotting enzyme and factor C, mammalian blood coagulation factors (activated protein C, factor Xa and alpha-thrombin), papain, and thermolysin. The complete amino acid sequence of this inhibitor was determined and its sequence was compared with those of bovine pancreatic trypsin inhibitor (BPTI) and other Kunitz-type inhibitors. It was found that the amino acid sequence of this inhibitor has a high homology of 47 and 43% with those of sea anemone inhibitor 5-II and BPTI, respectively. Thus, this protease inhibitor appeared to be one of the typical Kunitz-type protease inhibitors.     

Protease II from Escherichia coli. Purification and characterization.

We have previously demonstrated the existence of two types of endopeptidase in Escherichia coli. A purification procedure is described for one of these, designated protease II. It has been purified about 13,500-fold with a recovery of 24%. The isolated enzyme appears homogeneous by electrophoresis and gel filtration. Its molecular weight is estimated by three different methods to be about 58,000. Its optimal pH is around 8. Protease II activity is unaffected by chelating agents and sulfhydryl reagents. Amidase and proteolytic activities are stimulated by calcium ion, which decreases the enzyme stability. Like pancreatic trypsin, this endopeptidase catalyses the hydrolysis of alpha-amino-substituted lysine and arginine esters. It appears distinct from the previously isolated protease I, which is a chymotrypsin-like enzyme. The apparent Michaelis constant for hydrolysis of N-benzoyl-L-arginine ethyl ester is 4.7 X 10(-4) M. The esterase activity is inhibited by diisopryopylphosphorofluoridate (Ki(app) equals 2.7 X 10(-3) M) and tosyl lysine chloromethyl ketone (Ki(app) equals 1.8 X 10(-5) M), indicating that serine and histidine residues may be present in the active site. However, protease II is insensitive to phenylmethanesulfonyl fluoride and several natural trypsin inhibitors. Its amidase and esterase activities are competitively inhibited by free arginine and aromatic amidines. The proteolytic activity measured on axocasein is very low. In contrast to trypsin, protease II is without effect on native beta-galactosidase. It easily degrades aspartokinase I and III. Nevertheless both enzymes are resistant to proteolysis in the presence of their respective allosteric effectors. These results provide further evidence that such differences in protease susceptibility can be related to the conformational state of the substrate. The possible implication of structural changes in the mechanism of preferential proteolysis in vivo, is discussed.     

Properties of p-nitrophenyl phosphatase activity from porcine neutrophils

p-nitrophenyl phosphatase activity is high in porcine neutrophils and was found in plasma membrane and granule fractions isolated from sucrose density gradients after nitrogen cavitation to disrupt the cells. Very little activity was found in the cytosol. The enzyme has optimum activity at alkaline pHs with a pH optimum of 10.3. The pH profile was fairly broad with activity still remaining at physiological pH. Orthovanadate was shown to be a potent competitive inhibitor of the enzyme with a Ki of 14 microM. Phosphate also inhibited but at millimolar concentrations and the two inhibitors bind in a mutually exclusive fashion. Evidence from experiments using divalent ion chelators and zinc ions suggested that the phosphatase is a zinc metalloenzyme. Beryllium was found to be a very potent, non-competitive inhibitor of the neutrophil enzyme (Ki = 1.1 microM). Levamisole and theophylline were both shown to be uncompetitive inhibitors of the porcine phosphatase (Ki = 0.2 mM and 1.2 mM respectively). The neutrophil phosphatase was inhibited by L-homoarginine but unaffected by L-phenylalanine and L-glutamate.     

Excess zinc ions are a competitive inhibitor for carboxypeptidase A

The mechanism for inhibition of enzyme activity by excess zinc ions has been studied by kinetic and equilibrium dialysis methods at pH 8.2, I = 0.5 M. With carboxypeptidase A (bovine pancreas), peptide (carbobenzoxyglycyl-L-phenylalanine and hippuryl-L-phenylalanine) and ester (hippuryl-L-phenyl lactate) substrates were inhibited competitively by excess zinc ions. The Ki values for excess zinc ions with carboxypeptidase A at pH 8.2 are all similar [Ki = (5.2-2.6) X 10(-5) M]. The apparent constant for dissociation of excess zinc ions from carboxypeptidase A was also obtained by equilibrium dialysis at pH 8.2 and was 2.4 X 10(-5) M, very close to the Ki values above. With arsanilazotyrosine-248 carboxypeptidase A ([(Azo-CPD)Zn]), hippuryl-L-phenylalanine, carbobenzoxyglycyl-L-phenylalanine, and hippuryl-L-phenyl lactate were also inhibited with a competitive pattern by excess zinc ions, and the Ki values were (3.0-3.5) X 10(-5) M. The apparent constant for dissociation of excess zinc ions from arsanilazotyrosine-248 carboxypeptidase A, which was obtained from absorption changes at 510 nm, was 3.2 X 10(-5) M and is similar to the Ki values for [(Azo-CPD)Zn]. The apparent dissociation and inhibition constants, which were obtained by inhibition of enzyme activity and spectrophotometric and equilibrium dialysis methods with native carboxypeptidase A and arsanilazotyrosine-248 carboxypeptidase A, were almost the same. This agreement between the apparent dissociation and inhibition constants indicates that the zinc binding to the enzymes directly relates to the inhibition of enzyme activity by excess zinc ions. Excess zinc ions were competitive inhibitors for both peptide and ester substrates.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of bovine hepatic fructose-1,6-diphosphatase by substrate analogs.

Purified bovine hepatic fructose-1,6-diphosphatase, which exhibits maximal activity at neutral pH, is competitively inhibited by several analogs of its substrate, fructose 1,6-diphosphate. These include glucose 1,6-diphosphate (Ki = 9.4 X 10(-5) M), hexitol 1,6-diphosphate (Ki = 2.3 X 10(-4) M), and 2,5-anhydro-D-mannitol 1,6-diphosphate (Ki = 3.3 X 10(-8) M), and 2,5-anhydro-D-glucitol 1,6-diphosphate (Ki = 5.5 X 10(-7) M). The Ki values for both 2,5-anhydro-D-mannitol 1,6-diphosphate and 2,5-anhydro-D-glucitol 1,6-diphosphate are lower than the Km of 1.4 X 10(-6) M for fructose 1,6-diphosphate. Since 2,5-anhydro-D-mannitol 1,6-diphosphate is an analog of the beta anomer of fructose 1,6-diphosphate and 2,5-anhydro-D-glucitol 1,6-diphosphate is an analog of the alpha anomer, the lower Ki for the mannitol analog may indicate that the beta anomer of fructose 1,6-diphosphate, which predominates in solution, is the true substrate. The substrate analog 1,5-pentanediol diphosphate inhibits slightly (K0.5 = 5 X 10(-3) M), but 1,4-cyclohexyldiol diphosphate does not. The Ki for product inhibition by sodium phosphate is 9.4 X 10(-3) M. 2,5-Anhydro-D-mannitol 1,6-diphosphate and alpha-D-glucose 1,6-diphosphate are substrates at pH 9.0, but not at pH 6.5.     

Investigations of the esterase, phosphatase, and sulfatase activities of the cytosolic mammalian carbonic anhydrase isoforms I, II, and XIII with 4-nitrophenyl esters as substrates

The esterase, phosphatase, and sulfatase activities of carbonic anhydrase (CA, EC 4.2.1.1) isozymes, CA I, II, and XIII with 4-nitrophenyl esters as substrates was investigated. These enzymes show esterase activity with 4-nitrophenyl acetate as substrate, with second order rate constants in the range of 753-7706M(-1)s(-1), being less effective as phosphatases (k(cat)/K(M) in the range of 14.89-1374.40M(-1)s(-1)) and totally ineffective sulfatases. The esterase/phosphatase activities were inhibited by sulfonamide CA inhibitors, proving that the zinc-hydroxide mechanism responsible for the CO(2) hydrase activities of CAs is also responsible for their esterase/phosphatase activity. CA XIII was the most effective esterase and phosphatase. CA XIII might catalyze other physiological reactions than CO(2) hydration, based on its relevant phosphatase activity.     

Triiodothyronamine--a beta-adrenergic metabolite of triiodothyronine?

Triiodothyronamine (Triam) is a potential metabolite of triidothyronine (T3), resulting from decarboxylation of the side-chain. In an attempt to elucidate the physiological properties of Triam we have investigated the binding of Triam to beta-adrenergic receptors, using turkey-erythrocytes and performing binding studies with ( (-)(3H)-dihydroalprenolol) ( (-)(3H)-DHA) as a specific beta-adrenergic ligand. The inhibition constant Ki for Triam was determined as 5 X 10(-6) M, compared to dopamine (Ki = 1,3 X 10(-2) M), norepinephrine (Ki = 3 X 10(-4) M), epinephrine (Ki = 5 X 10(-5) M) and isoproterenol (Ki = 3 X 10(-6) M). The inhibition of ( (-)(3H)-DHA)-binding by Triam was further compared with other iodothyronines thyroxine (T4), T3, 3,3',5'-triiodothyronine (rT3) and 3,3'-diiodothyronine (3,3'-T2). It is concluded that Triam binds to beta-adrenergic receptors like naturally occurring amines but different from typical circulating iodothyronines.     

Partial purification and characterization of phosphotyrosyl-protein phosphatase from Ehrlich ascites tumor cells

We have previously described a phosphotyrosylprotein phosphatase in membrane vesicles from human epidermoid carcinoma A431 cells which is inhibited by micromolar concentration of Zn2+ and is insensitive to ethylenediaminetetraacetic acid (EDTA) and NaF [Brautigan, D. L., Bornstein, P., & Gallis, B. (1981) J. Biol. Chem. 256, 6519-6522]. Here we present the identification and partial purification of a similar enzyme from lysates of Ehrlich ascites tumor cells. the enzyme was purified by using diethylaminoethyl-Sephadex, Zn2+ affinity, and Sephadex G-75 chromatography. During purification, the phosphatase was separated into at least three fractions, all of which exhibited very similar properties and an apparent molecular weight of 40 000 upon gel filtration. The enzyme dephosphorylated phosphotyrosine (P-Tyr)-containing carboxymethylated and succinylated (CM-SC) phosphorylase with an apparent Km of 0.8 microM, as well as P-Tyr containing casein and epidermal growth factor (EGF) receptor kinase, but did not dephosphorylate P-Ser-phosphorylase. The phosphatase was inhibited by Zn2+ at micromolar concentrations (K0.5 with EGF receptor kinase = 5 X 10(-6) M; with CM-SC phosphorylase = 3.3 X 10(-5) M) but not by millimolar concentrations of EDTA and NaF. No inhibition was seen with 1 mM tetramisole, a specific inhibitor of alkaline phosphatases. P-Tyr inhibited the enzyme by 50% at 0.4 X 10(-3) M, while Tyr, Pi, PPi, and p-nitrophenyl phosphate, an excellent substrate for alkaline phosphatases and structurally very similar to P-Tyr, exerted partial inhibition at concentrations above 10(-3) M. The pH optimum was found to be 6.5-7, depending on the substrate used. Very little activity was seen below pH 5 and above pH 8.5. These properties clearly distinguish this enzyme from alkaline phosphatases, as well as the neutral and acidic protein phosphatases so far described, and therefore define it as a new enzyme of the phosphatase family--a phosphotyrosyl-protein phosphatase.     

Natural killer cell cytolytic granule-associated enzymes. I. Purification, characterization, and analysis of function of an enzyme with sulfatase activity

An enzyme with sulfatase activity has been isolated from the granules of a rat NK leukemia cell line, CRNK-16. The enzyme has been purified from crude preparation, with a specific activity of 52 nmol/min/mg of protein, by DEAE ion exchange and Con A-Sepharose affinity chromatography, resulting in a specific activity of 230 nmol/min/mg of protein. The molecular mass of the purified enzyme was estimated to be 40 kDa by gel filtration chromatography at pH 7.4, but the enzyme had the ability to complex to molecular masses of greater than 300 kDa at low pH when crude granule extract was used as the starting sample, suggesting that it associates with other granule components. The enzyme was determined to be an arylsulfatase by its ability to (a) hydrolyze p-nitrophenyl sulfate (Km = 26.0 mM) and p-nitrocatechol sulfate (pNC sulfate) (Km = 1.1 mM) and (b) be inhibited by sulfite (Ki = 6.0 x 10(-7) M), sulfate (Ki = 1 x 10(-3) M), and phosphate (Ki = 4 x 10(-5) M) in a competitive manner. The pH optimum for enzymatic activity was determined to be 5.6. The role of this enzyme in cytolytic function was investigated by examining the effect of its substrates and inhibitors on granule- and cell-mediated lysis. pNC sulfate was shown to cause a dose-dependent inhibition of target cell lysis by isolated cytolytic granules (complete inhibition at 12.5 mM). Sulfite induced an incomplete inhibition (50% at 1 mM), whereas phosphate was essentially without inhibitory effect. Sulfate, on the other hand, altered lytic activity in a biphasic manner, inasmuch as it induced an inhibition of lysis at high concentrations and an increase of lysis at low concentrations. Cell-mediated lysis was inhibited by pNC sulfate in a dose-dependent fashion at concentrations greater than 2.5 mM, with nearly complete inhibition at 50 mM. Sulfate also altered the lytic activity by intact cells in a biphasic manner, although the effect was much less pronounced. Sulfite and phosphate caused only a 30% inhibition of lytic activity. These results suggest that the sulfatase enzyme is involved in NK cytolytic function, presumably at the lethal hit stage.     

Indomethacin inhibition of glutathione S-transferases

Indomethacin inhibited rat liver glutathione S-transferases (EC 2.5.1.18). Its inhibition was non-competitive with respect to 3,4-dichloronitrobenzene with an apparent Ki of 5.3 X 10(-5) M and uncompetitive with respect to glutathione with an apparent Ki of 4.0 X 10(-5) M. 4-Chlorobenzoic acid and 5-methoxy-2-methylindole-3-acetic acid, two metabolites of indomethacin, were weak inhibitors of the enzymes. On the other hand, meclofenamic acid was a competitive inhibitor of the enzymes with an apparent Ki of 3.0 X 10(-4) M. Possible significance of these findings in arachidonic acid metabolism is discussed.     

Deuterium nuclear magnetic resonance study of the interaction of substrates and inhibitors with soybean lipoxygenase

Deuterium NMR spectra for a series of selectively deuterated substrates and inhibitors in the presence of lipoxygenase-1 (EC 1.13.11.12) are presented. Extrapolation of the 2H NMR line widths yielded transverse relaxation rates for the bound inhibitors [2H21]dodecanoic acid (protonated at the 2,2-position), [2,2-2H]dodecanoic acid, and [12,12,12-2H]dodecanoic acid which are 1/T2,bd = 5.0 X 10(3), 1.12 X 10(4), and 1.16 X 10(3) s-1, respectively. The substrates [9,10,12,13-2H]linoleic acid and [11,11-2H]linoleic acid had 1/T2,bd = 8.2 X 10(3) and 7.95 X 10(3) s-1, respectively. Kinetic measurements established Ki = 1.5 X 10(-3) M for dodecanoic acid (lauric acid) inhibition of lipoxygenase when the substrate is linoleic acid (Km = 2.6 X 10(-5) M). Lipoxygenase, with Mr 102,000, is predicted to have a rotational correlation time tau c - 1.2 X 10(-7) s, yielding a 1/T2,bd = 1.56 X 10(4) s-1 for tightly bound ligand. Hence, the correlation times of the selectively deuterated inhibitors indicate internal motions are present in the bound species.     

Purification and characterization of phosphodiesterase from the venom of Trimeresurus mucrosquamatus

Phosphodiesterase was isolated from the venom of Trimeresurus mucrosquamatus from Taiwan using gel filtration on a Sephadex G-100 column, followed by anion or cation exchange chromatography. Phosphodiesterase was homogeneous as established by a single band on acrylamide gel electrophoresis and immunodiffusion. Phosphodiesterase activity was inhibited by ethylenediamine tetraacetic acid (EDTA), o-phenanthroline, thioglycolic acid or p-chloromercuribenzoate (PCMB) but not by soybean trypsin inhibitor (SBTI) or benzamidine. The molecular weight of this enzyme was determined to be approximately 140,000 and the isoelectric point was found to be pH 7.4 by isoelectric focusing with carrier ampholyte. The Michaelis constant (Km) of this enzyme for p-nitrophenyl thymidine-5'-phosphate and inhibition constant (Ki) for PCMB were found to be 5.6 X 10(-3) and 7.6 X 10(-4) M, respectively.     

Kinetics of inhibition of green crab (Scylla serrata) alkaline phosphatase by vanadate

Green crab (Scylla serrata) alkaline phosphatase is a metalloenzyme that catalyzes the nonspecific hydrolysis of phosphate monoesters. The kinetics of inhibition of the enzyme by vanadate has been studied. The time course of the hydrolysis of p-nitrophenyl phosphate catalyzed by the enzyme in the presence of different Na3VO4 concentrations showed that, at each Na3VO4 concentration, the rate decreased with increasing time until a straight line was approached, the slopes of the straight lines being the same for all concentrations. The results suggest that the inhibition of the enzyme by Na3VO4 is a slow, reversible reaction with fractional residual activity. The microscopic rate constants were determined for the reaction of the inhibitor with the enzyme. As compared with Na2HPO4 (Ki = 0.95 mM), Na2HAsO4 (Ki = 1.10 mM), and Na2WO4 (Ki = 1.55 mM), the results suggest that Na3VO4 (Ki = 0.135 mM) is a considerably more potent inhibitor than other inhibitors.     

Carbamate kinase of Lactobacillus buchneri NCDO110. I. Purification and properties

Carbamate kinase has been prepared from Lactobacillus buchneri NCDO110. An approximately 91-fold increase in the specific activity of the enzyme was achieved. The purified extract exhibited a single band following polyacrylamide gel electrophoresis. The apparent molecular weight as determined by gel electrophoresis was about 97,000. The enzyme is stable for 2 weeks at -20 degrees C. Maximum enzymatic activity was observed at 30 degrees C and pH 5.4 in 0.1 M acetate buffer. L. buchneri carbamate kinase requires Mg2+ or Mn2+; its activity is higher with Mn2+. The activation energy of the reaction was 4078 cal mol-1 for the reaction with Mn2+ and 3059 cal mol-1 for the reaction with Mg2+. From a Dixon plot a pK value of 4.8 was calculated. The apparent Km values for ADP with Mg2+ or Mn2+ were 0.71 X 10(-3) and 1.17 X 10(-3) M, respectively, and the apparent Km values for carbamyl phosphate with Mg2+ or Mn2+ were 1.63 X 10(-3) and 1.53 X 10(-3) M, respectively. ATP and CTP acted as inhibitors of this reaction and the following values were obtained: Ki (ATP)Mg2+ = 9.4 mM, Ki (ATP)Mn2+ = 6.2 mM, and Ki (CTP)Mg2+ = 4.4 mM.     

Thymidylate synthetase from Diplococcus pneumoniae, properties and inhibition by folate analogs.

Thymidilate synthetase (methylenetetrahydrofolate:dUMP C-methyltransferase) in crude extract from Diplococcus pneumoniae exhibits a partial but variable requirement for Mg-2+ depending upon the buffer. Optimum Mg-2+ concentration is between 0.014 and 0.02 M. The optimum pH for activity in a variety of buffers occurred as a broad peak between 7.0 and 7.7. In Tris/acetate buffer, but not in potassium phosphate buffer, the pH optimum was different in the presence and absence of Mg-2+. Methylation of uridylate, cytidylate and deoxycytidylate could not be demonstrated over a pH range of 5.0-8.0. The enzyme exhibited an apparent Km for deoxyuridylate of 3.08 - 10-5 M and an apparent Km for L-(+)(minus)-5,10-methylene tetrahydrofolate of 2.66 - 10-4 M. During molecular-sieve chromatography and sucrose density-gradient centrifugation, the enzyme was detectable only as a single catalytically active form of Mr 34 000-38 000. 2,4-Diamino quinazoline antifolates were better competitive inhibitors (Ki = 3-8 -10-6 M) of thymidylate synthetase than 2,4-diamino pteridines (Ki = 3- 10-5 M). 2-Amino-4-hydroxy-quinazolines were the best inhibitors (Ki = 1.3-2.9 - 10-6 M). All of the 2,4-diamino quinazolines and pteridines inhibited dihydrofolate reductase from D. pneumoniae in a nearly stoichiometric fashion (Ki = less than 10-10 M). The 2-amino-4-hydroxy-quinazolines were poor inhibitors of this enzyme (Ki = 10=5 M).     

Fluoro ketone inhibitors of hydrolytic enzymes

The use of fluoro ketones as inhibitors of hydrolytic enzymes has been investigated. The acetylcholine analogues 6,6-dimethyl-1,1,1-trifluoro-2-heptanone and 3,3-difluoro-6,6-dimethyl-2-heptanone are inhibitors of acetylcholinesterase with Ki values of 16 X 10(-9) M and 1.6 X 10(-9) M, respectively. These fluoro ketones are 10(4)-10(5) times better as inhibitors than the corresponding methyl ketone. Since nucleophiles readily add to fluoro ketones, it is likely that these compounds inhibit acetylcholinesterase by formation of a stable hemiketal with the active-site serine residue. Fluoro ketone substrate analogues are also inhibitors of zinc metallo- and aspartylproteases. 2-Benzyl-4-oxo-5,5,5-trifluoropentanoic acid is an inhibitor of carboxypeptidase A (Ki = 2 X 10(-7) M). Trifluoromethyl ketone dipeptide analogues are good inhibitors of angiotensin converting enzyme. An analogue of pepstatin that contains a difluorostatone residue in place of statine has been prepared and found to be an extremely potent inhibitor of pepsin (Ki = 6 X 10(-11) M). The hydrated ketones are probably the inhibitory species since they are structural mimics of the tetrahedral intermediate that forms during the hydrolysis of peptide substrates.     

Purfication and properties of a specific isoflavone 7-O-glucoside-6'-malonate malonyestrase from roots of chickpea (Cicer arietinum L.)

Protein extracts from roots of chickpea (Cicer arietinum L.) plants contained high esterase activity hydrolyzing malonate hemiesters of isoflavone 7-O-glucosides. Using 5,7-dihydroxy-4'-methoxyisoflavone (biochanin A) 7-O-glucoside-6"-malonate as a substrate, a specific malonylesterase was purified about 700-fold to near homogeneity. The purified enzyme possesses an extremely low enzyme activity with synthetic esterase substrates. Various putative nonspecific esterases, as tested with alpha-naphthylacetate, were removed during enzyme purification. The malonylesterase demonstrated a very high molecular mass in gel chromatography and in sedimentation analyses with sucrose gradients (greater than or equal to 2 X 10(6)). Analytical sodium dodecyl sulfate-polyacrylamide gel electrophoresis pointed to a single subunit of 32,000. The catalyzed reaction showed a pH optimum at 7.5 and a temperature optimum between 30 and 35 degrees C. The apparent Km for biochanin A 7-O-glucoside-6"-malonate was (4.2 +/- 1.2) X 10(-4) M. The malonylesterase was insensitive to the esterase inhibitors eserine and neostigmine (10(-3) M) as well as phenylmethylsulfonyl fluoride, paraoxon, and diisopropylfluorophosphate (10(-4) M). On the other hand enzyme activity was totally inhibited by Hg2+ ions (10(-5) M) and p-hydroxymercuribenzoate (10(-4) M), whereas iodoacetamide (10(-6)-10(-4) M) inhibited only partially. Di- and tricarboxylic acids strongly stimulated enzyme activity at 10(-2) M. These properties indicate that the malonylesterase from chickpea roots greatly differs from other known esterases. The possible biological function of the specific malonylesterase is discussed in relation to isoflavone conjugate metabolism in chickpea.     

Chemical replacement of P1' serine residue at the second reactive site of soybean protease inhibitor C-II

The P1' Ser(50) at the second reactive site of soybean protease inhibitor C-II was replaced with arginine to confirm the contribution of this residue to the inhibition. The Arg derivative had less trypsin inhibitory activity (Ki = 1 X 10(-7) M) than the Ser derivative (Ki = 2 X 10(-8) M), in contrast to the results obtained from studies on peanut protease inhibitor B-III reported in the previous paper (J. Biochem. 101, 723-728 (1987)). These results suggest that each Bowman-Birk type inhibitor has an amino acid at the P1' position inherently best suited to maintaining its inhibitory activity, and that serine is not unique for the P1' amino acid in Bowman-Birk type inhibitors.