Characterization of a 2',5'-oligoadenylate (2-5A)-dependent 37-kDa RNase L: azido photoaffinity labeling and 2-5A-dependent activation

Upregulation of key components of the 2',5'-oligoadenylate (2-5A) synthetase/RNase L pathway has been identified in extracts of peripheral blood mononuclear cells from individuals with chronic fatigue [corrected] syndrome, including the presence of a low molecular weight form of RNase L. In this study, analysis of 2',5'-Oligoadenylate (2-5A) binding and activation of the 80- and 37-kDa forms of RNase L has been completed utilizing photolabeling/immunoprecipitation and affinity assays, respectively. Saturation of photolabeling of the 80- and the 37-kDa RNase L with the 2-5A azido photoprobe, [(32)P]pApAp(8-azidoA), was achieved. Half-maximal photoinsertion of [(32)P]pApAp(8-azidoA) occurred at 3.7 x 10(-8) m for the 80-kDa RNase L and at 6.3 x 10(-8) m for the 37-kDa RNase L. Competition experiments using 100-fold excess unlabeled 2-5A photoaffinity probe, pApAp(8-azidoA), and authentic 2-5A (p(3)A(3)) resulted in complete protection against photolabeling, demonstrating that [(32)P]pApAp(8-azidoA) binds specifically to the 2-5A-binding site of the 80- and 37-kDa RNase L. The rate of RNA hydrolysis by the 37-kDa RNase L was three times faster than the 80-kDa RNase L. The data obtained from these 2-5A binding and 2-5A-dependent activation studies demonstrate the utility of [(32)P]pApAp(8-azidoA) for the detection of the 37-kDa RNase L in peripheral blood mononuclear cell extracts.     

Interaction of oligonucleotides and ATP with preparations of sIgA possessing protein kinase activity

Interaction of secretory immunoglobulins A of a varying degree of purity with oligonucleotides and ATP has been studied by the method of affinity modification. For this aim we used reactive derivatives of 32P-labeled deoxyoligonucleotide (ClRCH2NHp(T)14) and [gamma-32P]ATP (ClR-32PppA) or ATP (ClR-pppA) bearing a 4-[(N-2-chloroethyl-N-methyl)amino]benzylamine residue. Preparations of sIgA were obtained from human milk by sequential chromatography on protein A-sepharose (P1), DEAE-fractogel (P2) and by gel-filtration in 50 mM NaOH (P3). It was revealed, that the H- and L-chains of sIgA P1; H-, L-chains and secretory component (SC) in sIgA P2 and only SC in sIgA P3 were exposed to modification after incubation with ClRCH2NHp(T)14. LPS, DNA, tRNA, heparin, sufficiently inhibited the modification of chains of sIgA P1. These competitors did not influence the modification of H- and L-chains of sIgA P2, but DNA, tRNA, heparin, inhibited binding of SC with the modifier. Suppressing affect of binding of ClRCH2NHp(T)14 with secretory component of sIgA P3 by d(T)14 has been observed as well. The research of ClR-32PppA interaction with sIgA P3 has shown that H- and L-chains of sIgA are exposed to modification. ATP inhibited the reaction. Study of the influence of modification on the protein kinase activity of sIgA P3 has revealed, that the preliminary incubation of sIgA P3 with ClR-pppA leads to inhibition of protein kinase activity. We suggest that sIgA, possessing the protein kinase activity (sIgA-abzymes) has an ATP-binding center (catalytic center) and has an oligonucleotide-binding center as well.     

Production of 2-phenylethanol in roses as the dominant floral scent compound from L-phenylalanine by two key enzymes, a PLP-dependent decarboxylase and a phenylacetaldehyde reductase

We investigated the biosynthetic pathway for 2-phenylethanol, the dominant floral scent compound in roses, using enzyme assays. L-[(2)H8] Phenylalanine was converted to [(2)H8] phenylacetaldehyde and [(2)H8]-2-phenylethanol by two enzymes derived from the flower petals of R. 'Hoh-Jun,' these being identified as pyridoxal-5'-phosphate-dependent L-aromatic amino acid decarboxylase (AADC) and phenylacetaldehyde reductase (PAR). The activity of rose petal AADC to yield phenylacetaldehyde was nine times higher toward L-phenylalanine than toward its D-isomer, and this conversion was not inhibited by iproniazid, a specific inhibitor of monoamine oxidase. Under aerobic conditions, rose petal AADC stoichiometrically produced NH3 together with phenylacetaldehyde during the course of decarboxylation and oxidation, followed by the hydrolysis of L-phenylalanine. Phenylacetaldehyde was subsequently converted to 2-phenylethanol by the action of PAR. PAR showed specificity toward several volatile aldehydes.     

A novel ribonuclease with antiproliferative activity from fresh fruiting bodies of the edible mushroom Hypsizigus marmoreus

An 18-kDa ribonuclease (RNase) with a novel N-terminal sequence was purified from fresh fruiting bodies of the mushroom Hypsizigus marmoreus. The purification protocol comprised ion exchange chromatography on DEAE cellulose, affinity chromatography on Affi-gel blue gel, ion exchange chromatography on CM-cellulose and Q-Sepharose and gel filtration by fast protein liquid chromatography on Superdex 75. The starting buffer was 10 mM Tris-HCl buffer (pH 7.2), 10 mM Tris-HCl buffer (pH 7.2), 10 mM NH(4)OAc buffer (pH 5), 10 mM NH(4)HCO(3) buffer (pH 9.4) and 200 mM NH(4)HCO(3) (pH 8.5), respectively. Absorbed proteins were desorbed using NaCl added to the starting buffer. A 42-fold purification of the enzyme was achieved. The RNase was unadsorbed on DEAE cellulose, Affi-gel blue gel and CM-cellulose but adsorbed on Q-Sepharose. It exhibited maximal RNase activity at pH 5 and 70 degrees C. Some RNase activity was detectable at 100 degrees C. It demonstrated the highest ribonucleolytic activity (196 U/mg) toward poly C, the next highest activity (126 U/mg) toward poly A, and much weaker activity toward poly U (48 U/mg) and poly G (41 U/mg). The RNase inhibited [(3)H-methyl]-thymidine uptake by leukemia L1210 cells with an IC(50) of 60 microM.     

Differences in the patterns of methylation in rat liver ribosomal ribonucleic acid after reaction in vivo with methyl methanesulphonate and NN-dimethylnitrosamine

1. rRNA was isolated from rat liver at short intervals after the intraperitoneal injection of [(14)C]methyl methanesulphonate (50mg/kg) or NN-di[(14)C]methylnitrosamine (2mg/kg). These doses were chosen to minimize the effects of toxicity. 2. The following methods of hydrolysis of [(14)C]methylated rRNA were employed: enzymic digestion to nucleosides at pH8; alkaline hydrolysis and conversion into nucleosides; acid hydrolysis to bases. 3. The methylation products were analysed by chromatography on columns of Dowex-50 (H(+) form) and Dowex-50 (NH(4) (+) form). 4. With both methylating agents the principal product of methylation was 7-methylguanine. Differences were obtained, however, in the molar proportions of the minor bases 3-methylcytosine, 1-methyladenine and 7-methyladenine. Methylation at the O-6 position of guanine was a significant feature of rRNA obtained from the NN-di[(14)C]methylnitrosamine-treated animals but was not detected in rRNA after treatment with [(14)C]methyl methanesulphonate.     

Differential modulation by N4, 2'-O-dibutyryl cytidine 3':5'-cyclic monophosphate of neutrophil activation.

The cyclic pyrimidine nucleotide, cCMP, is an endogenous substance in mammalian cells but little is known on its functional role. We studied the effects of cCMP, its cell-permeant analogue, N4,2'-O-dibutyryl cytidine 3':5'-cyclic monophosphate (Bt2cCMP), and of butyrate on superoxide (O2-) formation and cytosolic Ca2+ [( Ca2+]i) in human neutrophils. Bt2cCMP inhibited O2- formation and the rise in [Ca2+]i induced by a chemotactic peptide at submaximally effective concentrations. O2- formation induced by platelet-activating factor was potentiated by Bt2cCMP, whereas the cyclic nucleotide had no effect on the rise in [Ca2+]i induced by this agonist. Bt2cCMP enhanced O2- formation induced by tau-hexachlorocyclohexane at a submaximally effective concentration. O2- formation stimulated by complement C5a, concanavalin A, NaF, A 23187, phorbol myristate acetate and arachidonic acid was not affected by Bt2cCMP. cCMP was less effective than Bt2cCMP to inhibit fMet-Leu-Phe-induced O2- formation, and butyrate was without effect on any of the functional parameters studied. Our data show that a cell-permeant analogue of cCMP differentially inhibits and potentiates activation of human neutrophils.     

Purification, kinetic and thermodynamic studies of a new ribonuclease from a mutant of Aspergillus niger

Ribonuclease was purified from Aspergillus niger SA-13-20 to homogeneity level by using (NH(4))(2)SO(4) precipitation, DEAE-cellulose anion-exchange chromatography, ultrafiltration and Sephacryl HR-200 chromatography. The molecular weight and isoelectric point of the enzyme was 40.1kDa and 5.3, respectively. The pH- and temperature-dependent kinetic parameters were determined. The RNase showed the strongest affinity with RNA as the substrate, and the highest catalytic efficiency for hydrolysis of the substrate at pH 3.5 and 65 degrees C. It exhibited Michaelis-Menten Kinetics with k(cat) of 118.1s(-1) and K(m) of 57.0 microg ml(-1), respectively. Thermodynamic parameters for catalysis and thermal denaturation were also determined. Activation energy (E(a)) for catalysis of A. niger SA-13-20 RNase was 50.31 kJ mol(-1) and free energy (DeltaG(#)), enthalpy (DeltaH(#)) and entropy (DeltaS(#)) of activation for catalysis of the enzyme at 65 degrees C were 69.76, 47.50 and -65.83 Jmol(-1)K(-1), respectively. Activation energy (E(a,d)) for denaturation of the enzyme was 200.53 kJ mol(-1) and free energy (DeltaG(d)(#)), enthalpy (DeltaH(d)(#)) and entropy (DeltaS(d)(#)) of activation for denaturation of the enzyme at 45 degrees C were 79.18 kJ mol(-1), 197.88 and 373.09 Jmol(-1)K(-1), respectively.     

Heparan sulphate sulphotransferase. Properties of an enzyme from ox lung

A heparan sulphate sulphotransferase was partially purified from an ox lung homogenate by (NH(4))(2)SO(4) precipitation. Various glycosaminoglycans were assayed as sulphate acceptors with this enzyme. The highest acceptor activity was obtained with desulphated heparin and heparan sulphate, which indicates that sulphate transfer may be to free amino groups of the substrate. Some heparan sulphate was (35)S-labelled by incubation with the enzyme and re-isolated. On treatment of this heparan [(35)S]sulphate with nitrous acid and separation of the degradation products on Sephadex G-15, a major peak of radioactivity was obtained, and identified as [(35)S]sulphate by high-voltage electrophoresis at pH5.3. The [(35)S]sulphate is believed to be derived from N-[(35)S]sulphated groups of heparan [(35)S]-sulphate. That the ox lung preparation contained an N-sulphotransferase was confirmed by the isolation of 2-deoxy-2-[(35)S]sulphoamino-d-glucose as the major product from the flavobacterial degradation of heparan [(35)S]sulphate.     

Studies of the kinetics of the isolated mitochondrial ATPase using dinitrophenol as a probe

1. Dinitrophenol and maleate anions increase VATP on the 'washed', isolated, mitochondrial ATPase. Hydrolyses of iso-GTP and 2'-deoxy ATP are also stimulated, while hydrolyses of other nucleoside triphosphates (ITP, GTP etc.) are not. 2. Preincubation with ATP, iso-GTP or 2'-deoxy ATP results in a metastable enzyme form with a raised V and a reduced Km. Dinitrophenol stimulates both ATP and ITP hydrolyses by this form. 3. The Arrhenius plot of ATP (but not ITP) hydrolysis by the isolated ATPase shows a break at about 18 degrees C, apparently because the rate limiting step of hydrolysis changes as the temperature rises. 4. Adenylyl beta, gamma-imidodiphosphate (AdoPP[NH]P) inhibits ITP hydrolysis in a pseudofirst order reaction. Its binding is competitive with ITP. If the enzyme is preincubated with ATP, the rate of AdoPP[NH]P binding increases. It is concluded that AdoPP[NH]P inhibits by binding to the hydrolytic site of the enzyme. 5. We conclude that ATP hydrolysis is limited by diphosphate release and ITP hydrolysis by bond splitting. Energy release during ATP hydrolysis is maximal at the ATP binding step, and during ITP hydrolysis at bond splitting.     

The phosphorylated intermediate in the phosphoglyceromutase reaction

1. High-voltage paper-electrophoresis methods have been used for the separation of (32)P-labelled phosphoesters. 2. Evidence is presented which indicates that (32)P-labelled phosphopeptides, obtained after acid hydrolysis of phosphoglyceromutase incubated with impure 2,3-di[(32)P]phosphoglycerate, are derived from phosphoglucomutase contamination. 3. The hydrolysis of 2,3-di[(32)P]phosphoglycerate by phosphoglyceromutase has been studied. After an apparent instantaneous hydrolysis of 1 mole of coenzyme/mole of enzyme the reaction proceeds at a very low rate. 4. This hydrolysis seems to be due to the destruction of an enzyme-coenzyme complex. The proportions of the decomposition products of the complex vary according to further handling (pH of ionophoresis). 5. The inorganic [(32)P]phosphate produced by the hydrolysis of the complex and the inorganic [(32)P]phosphate produced by the slow phosphatase activity can be differentiated by the ability of the former to be incorporated into non-radioactive substrate before enzyme denaturation. 6. The effect of enzyme concentration on the stoicheiometry of the slow phosphatase hydrolysis of the diphosphoglycerate is described and this suggests that it may occur via two independent reactions, one of them being the decomposition of the enzyme-coenzyme complex on standing.     

Transcription in Isolated Wheat Nuclei: I. ISOLATION OF NUCLEI AND ELIMINATION OF ENDOGENOUS RIBONUCLEASE ACTIVITY

Nuclei isolated from embryos of wheat (var. Yamhill) incorporated [(3)H]UTP into a trichloroacetic acid-insoluble product linearly for 60 minutes. When the RNA synthesized in vitro was analyzed on a sucrose gradient, the amount of RNA in the 4S region increased with longer incubation times. These data and the absence of higher molecular weight RNA of specific size classes in our work (and previously published reports) suggested that nuclear fractions from plant tissue contained active nucleases. This was confirmed when wheat nuclei were mixed with [(3)H]yeast RNA (4, 18, 26S). All of the radioactive yeast RNA was degraded within 30 minutes to species sedimenting between 4 and 10S. The inclusion of high salt (125 millimolar (NH(4))(2)SO(4), 100 millimolar KCl), EGTA, and exogenous RNA or DNA reduced but did not eliminate endogenous RNase activity. Wheat embryo nuclei were further purified by centrifugation on a gradient of a polyvinylpyrrolidone-coated colloidal silica suspension (Percoll). These nuclei were ellipsoidal, free of cytoplasmic material, and lacked endogenous nuclease activity when assayed with [(3)H]yeast RNA. Sucrose gradients were not as effective as Percoll gradients in purifying nuclei free of RNase activity. The Percoll method of isolating nuclei and the RNase assay reported here will be useful in isolating plant nuclei that are capable of synthesizing distinct RNA species in vitro.     

The effect of bases non-complementary to the template on the efficacy of primer interaction with the Klenow fragment of DNA polymerase I from Escherichia coli

The comparison of the Km and Vmax values for the primers was carried out. The primers were either completely complementary to the template or contained non-complementary bases at different positions with respect to the 3'-end. The addition of NaF, selectively inhibiting 3'----5'-exonuclease activity of the enzyme, was shown to result in the increase of Vmax values by 10% and 30% for complementary and partially complementary primers, respectively, Km values of the latters being unchanged. Km values for d[(pT)10pC] is about 146-fold greater than that for d[(pT)11]. Km values for d[(pT)7pC(pT)2] (20 microM) and d[[(pT)2pC]3pT] (20 microM); d[(pT)4pC(pT)5] (5.0 microM); d[(pC)(pT)7] (1.3 microM) and d[(pT)2pC(pT)7] (1.2 microM) are comparable with those for d[(pT)2] (22 microM), d[(pT)5] (4.1 microM) and d[(pT)7] (1.2 microM), respectively, but not with the decathymidylate d[(pT)10] (0.2 microM). We suggest that it is not the length of the primers but the number of bases in the fragment beginning with the first nucleotide from the 3'-end and ending in the non-complementary base, that determines the efficiency of interaction of the primers containing non-complementary bases with the enzyme. The addition of one link to d(pT)n (n less than or equal to 10) resulted in a 1.8-fold increase in the affinity. When 11 less than n less than 25 the affinity is decreased so that d(pT)22-23 have minimal affinity to the enzyme. The primers containing more than 50 units were found to have about the same affinity (calculated on base concentration) as d(pT)10-11.     

DNA-Dependent RNA Polymerase Activity Associated with Subviral Particles of Polyhedral Cytoplasmic Dexoyribovirus

Polyhedral cytoplasmic deoxyribovirus virions contain a DNA-dependent RNA polymerase which catalyzes the incorporation of ribonucleotides into an acid-precipitable product. Treatment of virions with sodium deoxycholate and dithiothreitol resulted in the formation of subviral particles which could be separated from virions by rate zonal centrifugation in sucrose gradients. Subviral particles were RNA polymerase-positive and more active per unit mass of protein than virions. In vitro enzyme activity associated with subviral particles required addition of ribonucleotides, Mg(2+), and exogenous denatured DNA template. Optimal enzyme activity occurred over a broad pH (7.2 to 8.8) and Mg(2+) concentration (2 to 10 mumol) range. The specific activity of the RNA polymerase was maximal at 37 C. Addition of DNase or actinomycin D to the reaction mixture reduced the incorporation of [(3)H]UMP into an acid-precipitable product. The product of the reaction was sensitive to degradation by RNase but not to DNase or Pronase. These data suggest that the enzyme copies DNA into RNA.     

PDE7A1 hydrolyzes cCMP

The degradation and biological role of the cyclic pyrimidine nucleotide cCMP is largely elusive. We investigated nucleoside 3′,5′-cyclic monophosphate (cNMP) specificity of six different recombinant phosphodiesterases (PDEs) by using a highly-sensitive HPLC–MS/MS detection method. PDE7A1 was the only enzyme that hydrolyzed significant amounts of cCMP. Enzyme kinetic studies using purified GST-tagged truncated PDE7A1 revealed a cCMP K_M value of 135 ± 19 μM. The V_max for cCMP hydrolysis reached 745 ± 27 nmol/(min mg), which is about 6-fold higher than the corresponding velocity for adenosine 3′,5′-cyclic monophosphate (cAMP) degradation. In summary, PDE7A is a high-speed and low-affinity PDE for cCMP.     

Effects of ATP and monovalent cations on Mg2+ inhibition of (Na,K)-ATPase

The hydrolysis of ATP catalyzed by purified (Na,K)-ATPase from pig kidney was more sensitive to Mg2+ inhibition when measured in the presence of saturating Na+ and K+ concentrations [(Na,K)-ATPase] than in the presence of Na+ alone, either at saturating [(Na,Na)-ATPase] or limiting [(Na,0)-ATPase] Na+ concentrations. This was observed at two extreme concentrations of ATP (3 mM where the low-affinity site is involved and 3 microM where only the catalytic site is relevant), although Mg2+ inhibition was higher at low ATP concentration. In the case of (Na,Na)-ATPase activity, inhibition was barely observed even at 10 mM free Mg2+ when ATP was 3 mM. When (Na,K)-ATPase activity was measured at different fixed K+ concentrations the apparent Ki for Mg2+ inhibition was lower at higher monovalent cation concentration. When K+ was replaced by its congeners (Rb+, NH+4, Li+), Mg2+ inhibition was more pronounced in those cases in which the dephosphorylating cation forms a tighter enzyme-cation complex after dephosphorylation. This effect was independent of the ATP concentration, although inhibition was more marked at lower ATP for all the dephosphorylating cations. The K0.5 for ATP activation at its low-affinity site, when measured in the presence of different dephosphorylating cations, increased following the sequence Rb+ greater than K+ greater than NH+4 greater than Li+ greater than none. The K0.5 values were lower with 0.05 mM than with 10 mM free Mg2+ but the order was not modified. The trypsin inactivation pattern of (Na,K)-ATPase indicated that Mg2+ kept the enzyme in an E1 state. Addition of K+ changed the inactivation into that observed with the E2 enzyme form. On the other hand, K+ kept the enzyme in an E2 state and addition of Mg2+ changed it to an E1 form. The K0.5 for KCl-induced E1-to-E2 transformation (observed by trypsin inactivation profile) in the presence of 3 mM MgCl2 was about 0.9 mM. These results concur with two mechanisms for free Mg2+ inhibition of (Na,K)-ATPase: "product" and dead-end. The first would result from Mg2+ interaction with the enzyme in the E2(K) occluded state whereas the second would be brought about by a Mg2+-enzyme complex with the enzyme in an E1 state.     

Preparation of the bifunctional enzyme ribonuclease-deoxyribonuclease by cross-linkage.

Protease-free bovine pancreatic deoxyribonuclease (DNase) (1.6 X 10(-4) mmol) was thiolated on the NH2 groups with N-acetyl-DL-homocysteine thiolactone (2.4 X 10(-2) mmol) at pH 10.5 with imidazole (2.4 X 10(-2) mmol) as the catalyst in the presence of 4,4'-dithiodipyridine (4.2 X 10(-2) mmol). The product obtained after 16 h at 4 degrees C, 2-acetamido-4-(4'-dithiopyridyl)butyryl-DNase, isolated by gel filtration, contained an average of 0.87 +/- 0.13 mol of mixed disulfide per mol of DNase. Ribonuclease (RNase) was thiolated in a similar manner, but under N2 in the absence of 4,4'-dithiodipyridine. The protein N-acetylhomocysteinyl-RNase contained on the average 0.94 +/- 0.11 mol of sulfhydryl groups per mol of RNase. The coupling of RNase ot DNase was accomplished by thiol-disulfide interchange at pH 6.2 and 25 degrees C for 90 min. The hybrid enzyme (yield 25--33%, based upon the DNase derivative used) was freed from unreacted DNase, RNase, and homodimers by gel filtration, affinity chromatography, and salting-out chromatography. The purified enzyme contained one molecule each of DNase and RNase and hydrolyzed thymus deoxyribonucleic acid (DNA) and yeast or transfer ribonucleic acid (RNA) with 75 and 40% of the efficiencies, respectively, of the parent enzymes. The RNA strand of the hybrid substrate, phage f1 DNA-[3H]RNA, prepared from phage DNA with RNA polymerase, was hydrolyzed rapidly by the hybrid enzyme but was not hydrolyzed by RNase alone. A conjugate of the two enzymes offers the possibility in vivo of delivering two enzymes that differ in size, charge, and biological function to the same site at the same time.     

Crystallographic study of mechanism of ribonuclease T1-catalysed specific RNA hydrolysis

Ribonuclease T1 (RNase T1) cleaves the phosphodiester bond of RNA specifically at the 3'-end of guanosine. 2'-guanosinemonophosphate (2'-GMP) acts as inhibitor for this reaction and was cocrystallized with RNase T1. X-Ray analysis provided insight in the geometry of the active site and in the parts of the enzyme involved in the recognition of guanosine. RNase T1 is globular in shape and consists of a 4.5 turns alpha-helix lying "below" a four-stranded antiparallel beta-sheet containing recognition center as well as active site. The latter is indicated by the position of phosphate and sugar residues of 2'-GMP and shows that Glu58, His92 and Arg77 are active in phosphodiester hydrolysis. Guanine is recognized by a stretch of protein from Tyr42 to Tyr45. Residues involved in recognition are peptide NH and C = O, guanine O6 and N1H which form hydrogen bonds and a stacking interaction of Tyr45 on guanine. Although, on a theoretical basis, many specific amino acid-guanine interactions are possible, none is employed in the RNase T1.guanine recognition.     

Allosteric activation of the hydrolysis of specific substrates by chymotrypsin.

A variety of azobenzene compounds having bis-quaternary nitrogens have been shown to accelerate the hydrolysis by chymotrypsin of certain specific substrates by an allosteric mechanism. One of the most potent, 2,2'-bis[alpha-(benzyldimethylammonium)methyl]azobenzene dibromide (2,2'-QBzl) accelerated the hydrolysis of glutaryl-L-phenylalanine p-nitroanilide 40-fold at saturating concentration. Acceleration was by increasing kcat without altering Km. The hydrolysis of acetyl-L-tyrosine p-nitroanilide and acetyl-L-tyrosine anilide was also accelerated by Q-Bzl (25-fold and 1.8-fold respectively) while the hydrolysis of hemoglobin, azocoll and a number of esters was not affected. The inactivation of chymotrypsin by diphenylcarbamyl chloride and diphenylcarbamyl fluoride was accelerated by 2,2'-Q-Bzl. Reac;ivation in the presence of NH2OH was also accelerated, but in the absence of added nucleophile (i.e. of NH20H) no increase in rate was detectable. An allosteric effector was covalently attached to chymotrypsinogen A by reaction with 2,2'-bis[alpha-(o-bromomethylbenzyldimethylammonium)methyl]azobenezene dibromide. The product, when converted to active enzyme, was about 4 times more active than chymotrypsin as a result of an increase in kcat of hydrolysis; Km was unaffected. The mechanism of the allosteric acceleration process is not known but, because for all of the substrates affected acylation of the enzyme is rate-limitimg, it is tentatively suggested that the effectors facilitate proton transfer to the leaving group by an inductive effect on the 'charge relay system'. Spectral studies indicate that the allosteric site is a portion of the enzyme with a polarity near that of water, possibly on the outside surface of the enzyme molecule.