The mechanisms of inhibition of anion exchange in human erythrocytes by 1-ethyl-3-[3-(trimethylammonio)propyl]carbodiimide

Treatment of human erythrocytes with the membrane-impermeant carbodiimide 1-ethyl-3-[3-(trimethylammonio)propyl]carbodiimide (ETC) in citrate-buffered sucrose leads to irreversible inhibition of phosphate-chloride exchange. The level of transport inhibition produced was dependent on the concentration of citrate present during treatment, with a maximum of approx. 60% inhibition. [14C]Citric acid was incorporated into Band 3 (Mr = 95,000) in proportion to the level of transport inhibition, reaching a maximum stoichiometry of 0.7 mol citrate per mol Band 3. The citrate label was localized to a 17 kDa transmembrane fragment of the Band 3 polypeptide. Citrate incorporation was prevented by the transport inhibitors 4,4'-diisothiocyano- and 4,4'-dinitrostilbene-2,2'-disulfonate. ETC plus citrate treatment also dramatically reduced the covalent labeling of Band 3 by [3H]4,4'-diisothiocyano-2,2'-dihydrostilbene disulfonate (3H2DIDS). Noncovalent binding of stilbene disulfonates to modified Band 3 was retained, but with reduced affinity. We propose that the inhibition of anion exchange in this case is due to carbodiimide-activated citrate modification of a lysine residue in the stilbenedisulfonate binding site, forming a citrate-lysine adduct that has altered transport function. The evidence is consistent with the hypothesis that the modified residue may be Lys a, the lysine residue involved in the covalent reaction with H2DIDS. Treatment of erythrocytes with ETC in the absence of citrate resulted in inhibition of anion exchange that reversed upon prolonged incubation. This reversal was prevented by treatment in the presence of hydrophobic nucleophiles, including phenylalanine ethyl ester. Thus, inhibition of anion exchange by ETC in the absence of citrate appears to involve modification of a protein carboxyl residue(s) such that both the carbodiimide- and the nucleophile-adduct result in inhibition.     

Interaction of inhibitors with muscle phosphofructokinase.

The interaction of several inhibitors with muscle phosphofructokinase has been studied by both equilibrium binding measurements and kinetic analysis. At low concentrations of citrate a maximum of 1 mol is bound per mol of enzyme protomer. Tight binding requires MgATP and very weak binding is observed in the absence of either magnesium ion or ATP. ITP at low concentrations cannot replace ATP. In the presence of MgATP and at pH 7.0, the dissociation constant for the enzyme-citrate complex is 20 muM. At 50 muM citrate and excess magnesium ion, the concentration of ATP required to give half-maximal binding of citrate is approximately 3 muM . Both P-enolpyruvate and 3-P-glycerate compete for the binding of citrate and the estimated Ki values are 480 and 52 muM, respectively. Creatine-P, another inhibitor of muscle phosphofructokinase, does not compete with the binding of citrate. Measurement of the equilibrium binding of ATP shows that citrate, 3-P-glycerate, P-enolpyruvate, and creatine-P all increase the affinity of enzyme for MgATP with the concentration required to give an effect increasing in the order given. In kinetic studies, citrate, 3-P-glycerate and P-enolpyruvate each act synergistically with ATP to inhibit the phosphofructokinase reaction. This is indicated by the observation that the three metabolites do not inhibit the enzyme with ITP as the phosphoryl donor and that they inhibit at ATP concentrations that are not themselves inhibitory. Furthermore, the sensitivity to the inhibitors increases with increasing ATP concentrations. Striking differences in the extent of inhibition can be seen by varying the order of addition of assay components. Preincubation of the enzyme with ATP and citrate, 3-P-glycerate, or P-enolpyruvate results in greater inhibition than when the inhibitor is added after the reaction is started with fructose-6-P. Furthermore, the inhibition is reversed partially 10 to 15 min after the addition of fructose-6-P. This phenomenon is particularly striking with creatine-P as the inhibitor. Very high concentrations of this inhibitor are required to show any effect if the inhibitor is added after fructose-6-P. These effects are interpreted as reflecting slow conformational changes between an active form with high affinity for fructose-6-P and an inactive, or less active, conformation that binds the inhibitors. Citrate, 3-P-glycerate, P-enolpyruvate, and creatine-P increase the rate of the phosphofructokinase at subsaturating concentrations of MgITP. The results indicate a common binding site on the enzyme for citrate, 3-P-glycerate, and P-enolpyruvate that is distinct from the ATP inhibitory site. An additional site (or sites) for creatine-P is indicated. All four inhibitors act synergistically with ATP by increasing the affinity of the enzyme for MgATP at an inhibitory site. The inhibitors appear also to increase the affinity of the catalytic nucleoside triphosphate site for substrate.     

Chemical modification in situ of Escherichia coli 30 S ribosomal proteins by the site-specific reagent pyridoxal phosphate. Inactivation of the aminoacyl-tRNA and mRNA binding sites

epsilon-Amino groups of lysines of 30 S ribosomal subunits with affinity for phosphate groups were selectively modified in situ by reaction with pyridoxal phosphate and reduction of the Schiff base with nonradioactive or radioactive sodium borohydride. This reaction modified only a limited number of ribosomal proteins and resulted in the loss of only some 30 S activities. The modified proteins were identified and the extent of their modification determined. The main targets of the reaction were S3 greater than S1 greater than S6. The activity most severely affected by the pyridoxal phosphate reaction was mRNA-dependent aminoacyl-tRNA binding. Some inhibition of poly(U) binding was also observed, while neither binding of initiation factors nor association with 50 S subunits was inhibited. The inhibition of aminoacyl-tRNA binding showed distinct selectivity: the inhibition was far greater with NAcPhe-tRNA than with fMet-tRNA and with "A" site than with "P" site binding. In addition, initiation complex formation with some mRNAs (e.g. MS2 RNA) was affected more than with others (e.g. T7 early mRNA). Ribosome reconstitution experiments showed that the modification of protein S3 was the primary cause of the inhibition; a role was also played by ribosomal proteins S1, S2, and S21. Substrate protection experiments showed that the 30 S activity can be protected from pyridoxal phosphate inactivation upon formation of a ternary complex with poly(U) and tRNAPhe or NAcPhe-tRNAPhe. Accordingly, the extent of modification of ribosomal protein S3 was reduced in the ternary complex while modification of S1 was reduced in the presence of poly(U) alone.     

Site-specific modification of Escherichia coli DNA polymerase I large fragment with pyridoxal 5'-phosphate

Pyridoxal 5'-phosphate (PLP) is an inhibitor of DNA polymerase activity of Escherichia coli DNA polymerase I large fragment. Kinetic studies indicated that overall PLP inhibition was noncompetitive with respect to dNTP, and Hill plot analysis revealed that two molecules of PLP were involved in the inhibition. Reduction of the PLP-treated enzyme with sodium [3H]borohydride resulted in covalent incorporation of 3 mol of PLP/mol of enzyme. This incorporation was at lysine residues exclusively, and the PLP-modified enzyme was not capable of DNA polymerase activity. The presence of dNTP during the modification reaction blocked the incorporation of 1 mol of PLP/mol of enzyme. Similar results were obtained in the presence or absence of template-primer. These data indicate that a PLP target lysine is in or around a dNTP binding site that is essential for polymerase activity and that this binding site is functional in the absence of template-primer. The enzyme modified in the presence of dNTP, containing 2 mol of PLP/mol of enzyme, was capable of DNA polymerase activity but was unable to conduct elongation of product molecules beyond a short oligonucleotide length.     

Affinity labeling of the pyridoxal phosphate binding site of the beta2 subunit of Escherichia coli tryptophan synthase.

We have synthesized bromoacetylpyridoxamine phosphate and bromoacetylpyridoxamine and have shown that they meet three criteria for affinity labels of the beta2 subunit of tryptophan synthase: (i) the kinetic data of inactivation indicate that a binary complex is formed prior to covalent attachment; (ii) inactivation is largely prevented by the presence of pyridoxal phosphate; and (iii) inactivation is stoichiometric with incorporation of 0.7 to 0.8 mol of chromophore/mol of beta monomer. Our conclusion that inactivation of the apo beta2 subunit by bromoacetylpyridoxamine phosphate is due to the modification of cysteine is based on the disappearance of 1 mol of -SH/beta monomer and on the finding that [14C]carboxymethyl derivative in the acid hydrolysate of the protein modified by bromo[14C]acetylpyridixamine phosphate. A 39-residue tryptic peptide containing this essential cysteine has been isolated and purified from the bromo[14C]acetylpyridoxamine phosphate-labeled beta2 subunit.     

Rabbit muscle phosphofructokinase. 2. Inactivation by the affinity label 5'-[p-(fluorosulfonyl)benzoyl]-1,N6-ethenoadenosine

The reaction of the fluorescent affinity label 5'-[p-(fluorosulfonyl)benzoyl]-1,N6-ethenoadenosine with rabbit skeletal muscle phosphofructokinase results in an inactivation of the enzyme and in the covalent incorporation of up to one label/monomer. The substrates, MgATP and fructose 6-phosphate, each protect against inactivation of the enzyme, but neither diminishes the extent of covalent incorporation of the label, indicating that the inactivation is not the result of covalent incorporation of the label. Dithiothreitol reactivates the inactivated enzyme but does not reduce the extent of incorporation of the label. A determination of the number of free sulfhydryl groups on the enzyme as a function of the extent of inactivation by the reagent suggests that the inactivation is associated with the loss of two free sulfhydryl groups per phosphofructokinase monomer. The inactivation reaction appears to involve the reversible formation of an enzyme-reagent complex (Kd = 1.11 mM) prior to the conversion of the complex to inactive enzyme (k1 = 0.98 min-1). In view of the protection afforded by either substrate and the evidence suggesting the formation of an enzyme-reagent complex prior to inactivation, it would appear that the inactivation results from a reagent-mediated formation of a disulfide bond between two cysteinyl residues in close proximity, possibly in or near the catalytic site of the enzyme. The site of covalent attachment of the label appears to be the binding site specific for the activating adenine nucleotides cAMP, AMP, and ADP. The extent of covalent incorporation of the label at this site is diminished in the presence of cAMP, and phosphofructokinase modified at this site by this affinity label is no longer subject to activation by cAMP.     

Binding of regulatory ligands to rabbit muscle phosphofructokinase. A model for nucleotide binding as a function of temperature and pH.

The binding of nucleoside triphosphates to rabbit muscle phosphofructokinase has been determined in 0.05 M phosphate buffers by changes in intrinsic protein fluorescence and by direct binding measurements. These experiments have been performed over a wide range of pH, temperature, and effector concentration. Quenching of protein fluorescence is shown to measure binding of nucleotides to a site which is not the active site but rather a site responsible for inhibition of the kinetic activity. This site is relatively specific for either ATP or MgATP with free ATP binding about 10-fold more tightly than MgATP. A model to describe binding to this site as a function of pH and temperature is proposed. This model assumes that the apparent affinity for ATP is determined by protonation of two ionizable groups (per subunit) and that ATP binds exclusively to protonated enzyme forms. Several ligands which affect the apparent affinity for nucleotide binding at the inhibitory site act by shifting the apparent pK of the ionizable groups. NH4+ and citrate do not influence nucleotide binding to the inhibitory site. At pH 6.9 in 0.05 M phosphate, low concentrations of MgATP or MgGTP enhance the protein fluorescence due to binding at the active site. The fluorescence studies and direct binding studies show that there is one active site and one inhibitory site per subunit. As described elsewhere (Pettigrew, D. W., and Frieden, C. (1978) J. Biol. Chem. 253, 3623-3627), there is a third nucleotide binding site on each subunit which is specific for cAMP, AMP, and ADP.     

Binding of allosteric effectors to carbamyl-phosphate synthetase from Escherichia coli.

The binding of ornithine and inosine 5'-monophosphate (IMP), positive allosteric effectors, and of uridine 5'-monophosphate (UMP), a negative allosteric effector, to carbamyl-phosphate synthetase from Escherichia coli was studied by the technique of equilibrium dialysis. The monomeric form of the enzyme has one binding site for each of the three allosteric ligands. The binding of UMP is inhibited by ornithine, IMP, MgATP, and ammonia (also a positive allosteric effector). Bicarbonate, L-glutamine, and adenosine 5'-triphosphate (ATP) (Mg2+ absent) had no effect on the binding of UMP. The affinity of the enzyme for UMP was increased if phosphate buffer was replaced by 2-amino-2-hydroxymethyl-1,3-propanediol (Tris) buffer. The binding of ornithine was inhibited by UMP and ammonia, enhanced by MgATP, MgADP, and IMP, and not affected by bicarbonate, L-glutamine, or ATP (Mg2+ absent). Ornithine and ammonia probably bind to the same site on the enzyme. The binding of IMP is facilitated by ornithine and ammonia, but is inhibited by MgATP or ATP, indicating that adenine nucleotides can also bind to the IMP binding site. The results of these binding studies are consistent with a scheme previously proposed in which the allosteric effectors function by stabilizing one or the other of two different conformational states of the enzyme which are in equilibrium with each other (Anderson, P.M., and Marvin, S.V. (1970), Biochemistry 9, 171). According to this scheme, binding of the substrate MgATP is greatly facilitated when the enzyme exists in the conformational state stabilized by the positive allosteric effectors.     

Chemical modification of SH groups of E. coli phosphofructokinase-2 induces subunit dissociation: monomers are inactive but preserve ligand binding properties

Modification of Escherichia coli phosphofructokinase-2 (Pfk-2) with N-(1-pyrenil)maleimide results in an enzyme form that is inactive. However, the rate of modification is drastically reduced in the presence of the allosteric effector MgATP. The stoichiometry of the label incorporation was found to be 2.03 +/- 0.035 mol of the reagent/mol of subunit, in agreement with the number of titratable SH groups by 5,5'-dithiobis(2-nitrobenzoic acid) in the labeled protein. HPLC gel filtration experiments demonstrate that native Pfk-2 is a dimer in the absence of ligands, while in the presence of MgATP a dimer-tetramer transition is promoted. In contrast, the modified enzyme eluted as a monomer and the presence of MgATP was not able to induce aggregation. Although the modified monomers are inactive, the dissociation constants for the substrates and the allosteric effector MgATP, measured by following the fluorescence of the binding probe, are the same as for the native enzyme. Quenching of pyrene fluorescence emission of labeled phosphofructokinase-2 monomers by acrylamide gave downward curved Stern-Volmer plots, with very similar quenching efficiencies for the control and for the fructose-6-P and MgATP-enzyme complexes. These results show the presence of SH groups in the interface of Pfk-2 subunits, critical for subunit interactions, and that conformational changes occurring through the dimers are essential for catalytic activity.     

Amino acid sequence at the citrate allosteric site of rabbit muscle phosphofructokinase

Previously, this laboratory has demonstrated [Colombo, G., & Kemp, R. G. (1976) Biochemistry 15, 1774-1780] that under appropriate conditions the citrate inhibitory binding site of rabbit skeletal muscle phosphofructokinase can be covalently modified by using pyridoxal phosphate and sodium borohydride. In the current study, phosphofructokinase was modified by [3H]pyridoxal phosphate and sodium borohydride with or without the addition of citrate to protect the ligand binding site. The modified proteins were digested with trypsin, and the peptides were separated by high-pressure liquid chromatography. A comparison of the tryptic chromatographic profiles showed that while the label was broadly distributed among nine peaks in the elution profile of the enzyme modified in the presence of the protective ligand, a single peptide contained 70% of the total radioactivity of the enzyme modified in the absence of citrate. This peptide was presumed to contain at least part of the citrate inhibitory site of the enzyme. The sequence of the peptide was determined and shown to match with positions 528-536 of phosphofructokinase with the modified residue being Lys-529. A comparison of the sequence with that of procaryotic phosphofructokinase indicated that a homologous residue in the enzyme from Bacillus stearothermophilis is critical to an allosteric site. A second peptide that was the most abundant labeled peptide in the digest of the enzyme modified in the presence of citrate was found to be identical with the second most abundant peptide of the digest from the unprotected enzyme. This peptide corresponded to residues 681-692 with the lysine at position 684 being the site of phosphopyridoxylation.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of pyridoxal phosphate on the activity of aldolase from Lemna minor L.

Lemna aldolase has been purified by ion-exchange and affinity chromatography. The enzyme is inhibited by pyridoxal phosphate in a manner which suggests that pyridoxal phosphate forms a non-covalent complex with the enzymes which is in equilibrium with the Schiff base covalently modified enzyme. The kinetics of the reversal of inhibition have been used to test the proposition that the fall in aldolase activity observed during periods of nitrogen starvation is due to inhibition by pyridoxal phosphate. It is concluded that the in vivo loss of aldolase activity is not due to pyridoxal phosphate and that the in vitro inhibition of glycolytic enzymes by pyridoxal phosphate is due to the reaction with lysine residues at the active sites which are necessary to bind the strongly acidic sugar phosphates.     

Rabbit liver fructose 1,6-bisphosphatase: labeling of the active and allosteric sites with pyridoxal 5-phosphate and sequence of a nonapeptide from the active site

The active and allosteric sites of fructose 1,6-bisphosphatase (Fru-P₂ase, EC 3.1.3.11) were labeled by reaction with pyridoxal phosphate and sodium borohydride in the presence of the allosteric inhibitor AMP or the substrate, Fru-P₂, respectively. Modification of the active site results in loss of activity. Modification of the allosteric site decreases the sensitivity of the enzyme to inhibition by AMP and alters its ability to bind to blue dextran-Sepharose. The allosteric and active sites have been located on different cyanogen bromide peptides; the sequence of a nonapeptide from the active site is (H)GlyLysLeuArgLeuLeu TyrGluCys(OH). The lysyl residue is modified by pyridoxal phosphate.     

Identification and amino acid sequence of the deoxynucleoside triphosphate binding site in Escherichia coli DNA polymerase I

We have labeled the large fragment of Escherichia coli DNA polymerase I (Pol I) with pyridoxal 5'-phosphate, a substrate binding site directed reagent for DNA polymerases [Modak, M. J. (1976) Biochemistry 15, 3620-3626]. A covalent attachment of pyridoxal phosphate to Pol I results in the loss of substrate binding as well as the polymerase activity. The inactivation was found to be strictly dependent on the presence of a divalent metal ion. Four moles of pyridoxal phosphate was found to react per mole of the enzyme, while in the presence of substrate deoxynucleoside triphosphate only 3 mol of pyridoxal phosphate was bound. To identify the substrate-protected site on the enzyme, tryptic peptides from enzyme labeled with pyridoxal phosphate and tritiated borohydride, in the presence and absence of substrate, were resolved on a C-18 reverse-phase column. A single peptide containing the substrate-protected site was identified and further purified. The amino acid composition and sequence analysis of this peptide revealed it to span residues 756-775 in the primary acid sequence of Pol I. Lys-758 of this sequence was found to be the site of the pyridoxal phosphate reaction. It is therefore concluded that Lys-758 is the site of binding for the metal chelate form of nucleotide substrates in E. coli DNA polymerase I.     

Active-site modification of mammalian DNA polymerase beta with pyridoxal 5'-phosphate: mechanism of inhibition and identification of lysine 71 in the deoxynucleoside triphosphate binding pocket

Pyridoxal 5'-phosphate is a potent inhibitor of the DNA polymerase activity of recombinant rat DNA polymerase beta. Kinetic studies indicate that the mechanism of PLP inhibition is complex. In a lower range of PLP concentration, inhibition is competitive with respect to substrate dNTP, whereas at higher levels of PLP several forms of enzyme combine with PLP and are involved in the overall inhibition, and a possible model for these interactions during the catalytic process is suggested. Reduction of the PLP-treated enzyme with sodium [3H]borohydride results in covalent incorporation of about 4 mol of PLP/mol of enzyme, and the modified enzyme is not capable of DNA polymerase activity. The presence of dNTP during the modification reaction blocks incorporation of 1 mol of PLP/mol of enzyme, and the enzyme so modified is almost fully active. This protective effect is not observed in the absence of template-primer. Tryptic peptide mapping of the PLP-modified enzyme reveals four major sites of modification. Of these four sites, only one is protected by dNTP from pyridoxylation. Sequence analysis of the tryptic peptide corresponding to the protected site reveals that it spans residues 68-80 in the amino acid sequence of the enzyme, with Lys 71 as the site of pyridoxylation. These results indicate that Lys 71 is at or near the binding pocket for the dNTP substrate.     

Characterization of the ATP binding site on Escherichia coli DNA gyrase. Affinity labeling of Lys-103 and Lys-110 of the B subunit by pyridoxal 5'-diphospho-5'-adenosine

We have labeled the adenosine triphosphate binding site of Escherichia coli DNA gyrase with the ATP affinity analog, [3H]pyridoxal 5'-diphospho-5'-adenosine (PLP-AMP). PLP-AMP strongly inhibits the ATP-ase and DNA supercoiling activities of DNA gyrase, with 50% inhibition occurring at 7.5 microM inhibitor. ATP and ADP compete with PLP-AMP for binding and protect the enzyme against inhibition. The labeling appears to proceed by a Schiff base complex between the 4-formyl group of the pyridoxyl moiety of PLP-AMP and a protein primary amino group, since the inhibition and reagent labeling are reversible unless the complex is treated with NaBH4. Complete inactivation is estimated to occur upon the covalent incorporation of 2 mol of inhibitor/mol of gyrase. The Km for ATP was found to be unchanged for partially inhibited enzyme samples, suggesting an all-or-none type of inhibition. A 3H-labeled peptide spanning residues 93-131 of the B protein was isolated from a V-8 protease digest. Radioactive peaks corresponding to Lys-103 and Lys-110 were found during the Edman degradation, suggesting that these amino acids form part of the ATP binding site. A comparison of the amino acid sequence in this region with the sequences of other type II topoisomerases indicates the possible location of a common ATP binding domain.     

Labeling of specific lysine residues at the active site of glutamine synthetase

Glutamine synthetase (Escherichia coli) was incubated with three different reagents that react with lysine residues, viz. pyridoxal phosphate, 5'-p-fluorosulfonylbenzoyladenosine, and thiourea dioxide. The latter reagent reacts with the epsilon-nitrogen of lysine to produce homoarginine as shown by amino acid analysis, nmr, and mass spectral analysis of the products. A variety of differential labeling experiments were conducted with the above three reagents to label specific lysine residues. Thus pyridoxal phosphate was found to modify 2 lysine residues leading to an alteration of catalytic activity. At least 1 lysine residue has been reported previously to be modified by pyridoxal phosphate at the active site of glutamine synthetase (Whitley, E. J., and Ginsburg, A. (1978) J. Biol. Chem. 253, 7017-7025). By varying the pH and buffer, one or both residues could be modified. One of these lysine residues was associated with approximately 81% loss in activity after modification while modification of the second lysine residue led to complete inactivation of the enzyme. This second lysine was found to be the residue which reacted specifically with the ATP affinity label 5'-p-fluorosulfonylbenzoyladenosine. Lys-47 has been previously identified as the residue that reacts with this reagent (Pinkofsky, H. B., Ginsburg, A., Reardon, I., Heinrikson, R. L. (1984) J. Biol. Chem. 259, 9616-9622; Foster, W. B., Griffith, M. J., and Kingdon, H. S. (1981) J. Biol. Chem. 256, 882-886). Thiourea dioxide inactivated glutamine synthetase with total loss of activity and concomitant modification of a single lysine residue. The modified amino acid was identified as homoarginine by amino acid analysis. The lysine residue modified by thiourea dioxide was established by differential labeling experiments to be the same residue associated with the 81% partial loss of activity upon pyridoxal phosphate inactivation. Inactivation with either thiourea dioxide or pyridoxal phosphate did not affect ATP binding but glutamate binding was weakened. The glutamate site was implicated as the site of thiourea dioxide modification based on protection against inactivation by saturating levels of glutamate. Glutamate also protected against pyridoxal phosphate labeling of the lysine consistent with this residue being the common site of reaction with thiourea dioxide and pyridoxal phosphate.     

Mitochondrial nicotinamide nucleotide transhydrogenase: inhibition by ethoxyformic anhydride, dansyl chloride, and pyridoxal phosphate

Mitochondrial energy-linked nicotinamide nucleotide transhydrogenase (TH; EC 1.6.1.1) was inactivated by treatment with pyridoxal phosphate, ethoxyformic anhydride (EFA) or dansyl chloride. NADP and NADPH, but not NAD and NADH, protected TH against inhibition by pyridoxal phosphate, and L-lysine reversed this inhibition. The results suggested modification of an essential lysyl residue by pyridoxal phosphate, possibly at the NADP(H) binding site of TH. EFA and dansyl chloride inhibited TH in a similar manner. The effect of pH on the rate of inhibition of TH by EFA and dansyl chloride was the same, and in both cases addition of NADP and particularly NADPH accelerated the rate of inhibition, while addition of NAD or NADH had no effect. Double inhibition studies, using in one experiment dithiothreitol-reversible inhibition by 5,5'-dithiobis(2-nitrobenzoic acid) to protect the thiol groups of TH, and in another experiment lysine-reversible inhibition by pyridoxal phosphate to protect the putative essential lysyl residues of the enzyme, followed in each case by further treatment of the protected TH with EFA or dansyl chloride, suggested that the inhibitions by EFA and dansyl chloride were independent of the inhibitions by 5,5'-dithiobis (2-nitrobenzoic acid) and pyridoxal phosphate. The inhibitors discussed above are interesting, because pyridoxal phosphate is the only reagent known which appears to modify an essential residue in the NADP(H), but not the NAD(H), binding site of TH, and EFA and dansyl chloride are the only inhibitors known which appear to react with essential residues outside the active site of TH. It is possible that EFA and dansyl chloride inhibitions involve modification of essential prototropic residues in the proton translocation domain of the enzyme.     

Observations on the pyridoxal 5'-phosphate inhibition of DNA polymerases.

Pyridoxal 5'-phosphate at concentrations greater than 0.5 mM inhibits polymerization of deoxynucleoside triphosphate catalyzed by a variety of DNA polymerases. The requirement for a phosphate as well as aldehyde moiety of pyridoxal phosphate for inhibition to occur is clearly shown by the fact that neither pyridoxal nor pyridoxamine phosphate are effective inhibitors. Since the addition of nonenzyme protein or increasing the amount of template primer exerted no protective effect, there appears to be specific affinity between pyridoxal phosphate and polymerase protein. The deoxynucleoside triphosphates, however, could reverse the inhibition. The binding of pyridoxal 5'-phosphate to enzyme appears to be mediated through classical Schiff base formation between the pyridoxal phosphate and the free amino group(s) present at the active site of the polymerase protein. Kinetic studies indicate that inhibition by pyridoxal phosphate is competitive with respect to substrate deoxynucleoside triphosphate(s).     

Nonexistence of Exo-cellobiohydrolase (CBH) in the Cellulase System of Trichoderma viride

Chemical modification of histidine residues in ricin E was studied with regard to saccharide binding. The analytical data indicate that 6 out of 7 histidine residues in ricin E are eventually modified with diethylpyrocarbonate (DEP) at pH 6.0 and 25°C in the absence of specific saccharides. Modification of histidine residues greatly decreased the cytoagglutinating activity of ricin E, and only 10% of the residual activity was found after modification of 6 histidine residues/mol. The data of affinity chromatography using lactamyl- and galactosamine-cellulofine columns suggest that modification of histidine residues does not have much effect on the binding ability at the low affinity saccharide-binding site of ricin E but abolishes the binding ability at the high affinity saccharide-binding site. In the presence of lactose, one histidine residue/mol was protected from the DEP modification with retention of a fairly high cytoagglutinating activity. Such a protective effect was also observed for specific saccharides such as galactose and A^-acetylgalactosamine, but not for glucose, a non-specific saccharide. On treatment with hydroxylamine, the modified ricin E restored 67 % of the cytoagglutinating activity. Based on these findings, it is suggested that in the high affinity saccharide- binding site of ricin E there exists one histidine residue responsible for saccharide binding.     

Modification of Serratia marcescens anthranilate synthase with pyridoxal 5′-phosphate

The glutamine-dependent activity of Serratia marcescens anthranilate synthase was inactivated by pyridoxal 5′-phosphate and sodium cyanide. The reaction was specific in that the ammonia-dependent activity of the enzyme was unaffected. The inactivation was stable to dilution or dialysis but was reversed by dithiothreitol. The enzyme contains dissimilar subunits designated anthranilate synthase components I (AS I) and II (AS II). Incorporation of [¹⁴C]NaCN demonstrates that modification was limited to one to two residues per AS I · AS II protomer. An active site cysteine is involved in the glutamine-dependent activity. Modification by pyridoxal 5′-phosphate and NaCN blocked affinity labeling of the active site cysteine by the glutamine analog 6-diazo-5-oxo-l-norleucine and reduced alkylation of the active site cysteine by iodoacetamide. These results suggest modification is at the glutamine active site. Initial modification by iodoacetamide did not prevent pyridoxal 5′-phosphate-dependent incorporation of ¹⁴CN showing that the pyridoxal 5′-phosphate modification did not involve the essential cysteinyl residue. These results suggest that modification of a lysyl residue in the glutamine active site of anthranilate synthase reduces the reactivity of the essential cysteinyl residue resulting in the loss of the amidotransferase activity.