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PubMed1989

Isolation of a Staphylococcus aureus beta-lactamase-dicloxacillin complex and kinetic studies on the reactivation of the enzyme.

Exposure of the beta-lactamase from Staphylococcus aureus to the slowly reacting substrates cloxacillin or dicloxacillin results in time-dependent inactivation of the enzyme. Methods for the rapid separation of a beta-lactamase-dicloxacillin complex from excess inhibitor, using centrifuged columns of Sephadex G-25 or DEAE-Sephadex G-25, are described. The enzyme-dicloxacillin complex releases active enzyme, with specific activity identical to that of untreated enzyme, after storage at pH 7.5 at 15 degrees C. Full reactivation was accompanied by the release of 0.8 eq of hydrolyzed dicloxacillin. The complex is stable for up to 40 h when stored at pH 3 at 4 degrees C. The reactivation process, which occurs with first-order kinetics at 15 degrees C and pH values between 4 and 8, displays a pH dependence with apparent pKa's of 4.6 and 8.5, and a limiting value of the reactivation rate constant of 0.022 min-1. Deviation from first-order kinetics at pH 9 is consistent with a competing irreversible inactivation of the enzyme at that pH. This behavior differs substantially from that of the similarly inactivated beta-lactamase I from Bacillus cereus, whose rate of reactivation is independent of pH, but which undergoes irreversible denaturation at acidic pH [A. L. Fink, K. M. Behner, and A. K. Tan (1987) Biochemistry 26, 4248-4258]. Addition of hydroxylamine to the S. aureus beta-lactamase-dicloxacillin, complex stimulates the rate of reactivation by a maximum of 35%. This effect is hyperbolically dependent on the concentration of hydroxylamine with half-maximal stimulation at 2.8 mM. The Km for ampicillin hydrolysis catalyzed by the partially reactivated enzyme is identical to that measured for catalysis by the untreated enzyme. We discuss our observations in relation to models for the transient inhibition process.

Dicloxacillin↗
PubMed2006

Low background FRET-substrates for lipases and esterases suitable for high-throughput screening under basic (pH 11) conditions.

FRET-based fluorogenic substrates for lipases and esterases were prepared in four steps from commercially available building blocks. The substrates are pyrenebutyric acid monoesters of aliphatic 1,2-diols bearing a dinitrophenylamino group as a quencher. The most enzyme-reactive substrate is ester 2a. The substrates do not show any measurable background reaction in the absence of enzyme even at pH 11, but react fast and specifically with lipases and esterases. These substrates offer an unprecedented and practical solution to the long-standing problem of a simple yet efficient high-throughput screening tool for lipase activities under basic conditions.

Dinitrobenzenes↗
PubMed1980

Presence of two conformationally vicinal sulfhydryl groups at the active site of UDP-glucose 4-epimerase from Saccharomyces fragilis.

UDP-glucose 4-epimerase from Saccharomyces fragilis was inactivated by diazene dicarboxylic acid bis-N,N-dimethylamide or diamide, a compound that can specifically oxidize conformationally vicinal sulfhydryl groups on protein surfaces. The inactive enzyme was shown to retain the original dimeric structure and NAD, which is a coenzyme for this reaction, was not dissociated from the apoenzyme. The loss of activity was due to the direct modification of sulfhydryl groups and could not be attributed to any subsequent loss of structural integrity. The activity of the enzyme could be regained almost completely on incubation with mercaptoethanol alone and no exogenous NAD was needed for reactivation. The reactivated enzyme showed most of the characteristic properties of the native enzyme like activation by cations or inhibition by UMP. Presence of substrate provided partial protection against inactivation by the reagent. Formation of disulfide bond(s) across the subunits was demonstrated by sodium dodecyl sulfate gel electrophoresis in absence of mercaptoethanol. Titration of native and diamide-inactivated enzyme with p-chloromercuribenzoate revealed that only two sulfhydryl groups were involved in the formation of the disulfide cross-linkage across the subunits. The above results indicate the possible presence of two conformationally vicinal sulfhydryl groups at two different subunits of the enzyme that constitute part of the active site.

Binding Sites↗
PubMed1972

Thermal inactivation and reactivation of an enzyme in vivo. Pantothenate hydrolase of Pseudomonas fluorescens.

Thermal inactivation and reactivation of pantothenate hydrolase were studied in whole cells of Pseudomonas fluorescens. The enzyme is susceptible to thermal inactivation in whole cells at 37-40 degrees C, and is reactivated when the temperature is lowered again. Chloramphenicol does not prevent reactivation. The activation energy of enzyme inactivation in vivo is about 540kJ/mol. This activation energy is 220kJ/mol in vitro, but it is increased to 550-630kJ/mol by several metabolites, such as succinate, glyoxylate and oxalate. Generally, good carbon sources, causing rapid growth, protect the enzyme from thermal inactivation in vivo, and enable reactivation to occur at a fast rate. The enzyme is also inactivated below 35 degrees C, showing an activation energy of about 35kJ/mol. Good carbon sources prevent this inactivation as well, and cause slight reactivation. Glycine, although not utilized for growth, protects the enzyme well from this inactivation but not from inactivation at 37-40 degrees C, and prevents reactivation totally. From the activation energies of inactivation and the effects of the various carbon sources, it appears possible that changes in the concentrations of intracellular metabolites may be responsible for the changes in inactivation and reactivation.

Carbon Isotopes↗
PubMed1985

Reactivation of human placental 17 beta,20 alpha-hydroxysteroid dehydrogenase affinity alkylated by estrone 3-(bromoacetate): topographic studies with 16 alpha-(bromoacetoxy)estradiol 3-(methyl ether).

Estradiol 17 beta-dehydrogenase and 20 alpha-hydroxysteroid dehydrogenase, oxidoreductase activities copurified from the cytosol of human-term placenta as a homogeneous protein (native enzyme), were reactivated at equal rates to 100% activity following complete inactivation in the presence of cofactor (NADPH) with the affinity alkylator estrone 3-(bromoacetate). Reactivation was accomplished by base-catalyzed hydrolysis of steroidal ester-amino acid linkages in the enzyme active site. The rate of enzyme reactivation was pH dependent. In identical studies without NADPH, only 12% of the original enzyme activity was restored. Completely reactivated enzyme was repurified by dialysis. Enzyme in control mixtures (control enzyme) that contained estrone in place of alkylator was treated the same as the reactivated enzyme. Reactivated enzyme exhibited a 6.0-fold lower affinity for common substrates, a 1.8-fold lesser affinity for NAD+ and NADH, and the same affinity for NADP+ and NADPH compared to control enzyme. In incubations that included NADPH, the reactivated enzyme maintained full activity during a 20-h second exposure to estrone 3-(bromoacetate), but in identical incubations without NADPH, the reactivated enzyme was rapidly inactivated at the same rate as the control and native enzymes. The control and reactivated enzymes were inactivated at equal rates by 16 alpha-(bromoacetoxy)estradiol 3-(methyl ether) in the presence or absence of cofactor (NADP+) and exhibited similar Kitz and Wilson inhibition constants for this affinity alkylator. Estrone 3-(bromo[2'-14C]acetate) incubated with native enzyme and NADPH produced radiolabeled 3-(carboxymethyl)histidine and S-(carboxymethyl)cysteine.(ABSTRACT TRUNCATED AT 250 WORDS)

17-Hydroxysteroid Dehydrogenases↗
PubMed1996

Selegiline and lymphocyte superoxide dismutase activities in Parkinson's disease.

Superoxide dismutases (SODs) are metalloenzymes that detoxify superoxide radicals, and occur in cytosolic (Cu,Zn-SOD) and mitochondrial (Mn-SOD) forms in multiple tissues, including brain. A neuroprotective effect against oxide stressor exposures may be provided by SOD, although excessive enzyme activity can produce cell injury by formation of hydroxyl radical from hydrogen peroxide. We measured Cu,Zn-SOD and Mn-SOD activities in peripheral lymphocytes of 43 newly diagnosed idiopathic Parkinson's disease (PD) cases and 62 age- and sex-matched controls free of neurodegenerative disorders. Significant excesses of both SOD forms were found among PD cases compared with controls; however, the excesses were found exclusively among PD patients treated with the monoamine oxidase inhibitor selegiline (L-deprenyl). Enzyme-linked immunosorbent assays (ELISAs) confirmed that the activity excesses were due to increased protein rather than more highly reactive enzymes in lymphocytes of PD cases. Our findings clearly indicate the importance of selegiline on measured Cu,Zn-SOD and Mn-SOD activity in peripheral lymphocytes. Characterizing a possible therapeutic value of SOD will require longitudinal assessments of SOD in relation to PD progression.

Antiparkinson Agents↗
PubMed1997

Repair of oxidized DNA bases in the yeast Saccharomyces cerevisiae.

An essential requirement for all organisms is to maintain its genomic integrity. Failure to do so, in multicellular organisms such as man, can lead to degenerative pathologies such as cancer and aging. Indeed, a very low spontaneous mutation rate is observed in eukaryotes, suggesting either an inherent stability of the genome or efficient DNA repair mechanisms. In fact, DNA is subjected to unceasing attacks by a variety of endogenous and environmental reactive chemical species yielding a multiplicity of DNA damage, the deleterious action of which is counteracted by efficient repair enzymes. Reactive oxygen species formed in cell as by-products of normal metabolism are probably the major source of endogenous DNA damage. Amongst oxidative damage, base modifications constitute an important class of lesions whose lethal or mutagenic action has been established. Oxidatively damaged DNA bases are mostly repaired by the base excision repair pathway (BER) in prokaryotes and eukaryotes. However, the nucleotide excision repair pathway (NER) may also play a role in the repair of some oxidized bases in DNA. Here, we describe repair pathways implicated in the removal of oxidized bases in Saccharomyces cerevisiae. Yeast is a simple organism that can be used as a paradigm for DNA repair in all eukaryotic cells. S cerevisiae possesses three DNA glycosylases that catalyze the excision of oxidized bases from damaged DNA: the Ogg1, Ntg1 and Ntg2 proteins. The aim of this review is to summarize recent findings dealing with the formation, the biological consequences and the repair of oxidized DNA bases in S cerevisiae.

Amino Acid Sequence↗
PubMed1977

[Regulation of glutamine metabolism in Chlorella pyrenoidosa. Regulation of glutamine synthetase activity by adenylic system components].

A decrease of glutamine synthetase (E. C. 6.3.1.2.) activity was observed under the assimilation of ammonium nitrogen in Chlorella. At the same time a decrease of ATP content in Chlorella cells took place. The ATP content was 7-fold decreased, while ADP and AMP contents were 4-fold and 3-fold increased respectively, after 15 min. of Chlorella incubation on "ammonium" medium. Further incubation for 45 min, resulted in gradual increase of ATP content and in decrease of ADP and AMP contents. The value of energy charge in ammonium assimilating Chlorella cells sharply decreased for first 15 min. of incubation and then it normalized gradually. The experiments with glutamine synthetase preparation, isolated from ammonium assimilating cells, have shown that ADP and AMP are strong inhibitors of the enzyme in the presence of Mg2+, and only ADP produces the inhibitory effect in the presence of Mn2+. No enzyme reactivation was observed after the transfer of ammonium assimilating cells into nitrogen-free medium or nitrate medium, the enzyme activity increasing at the expense of enzyme protein synthesis denovo.

Adenine Nucleotides↗
PubMed1980

Variable enzymological patterning in tyrosine biosynthesis as a means of determining natural relatedness among the Pseudomonadaceae.

Enzymes of tyrosine biosynthesis (prephenate dehydrogenase and arogenate dehydrogenase) were characterized in 90 species currently classified within the genera Pseudomonas, Xanthomonas, and Alcaligenes. Variation in cofactor specificity and regulatory properties of the dehydrogenase proteins allowed the separation of five groups. Taxa defined by enzymological patterning corresponded strikingly with the five ribosomal ribonucleic acid (rRNA) homology groups established via rRNA-deoxyribonucleic acid hybridization. rRNA homology groups I, IV, and V all lack activity for arogenate/nicotinamide adenine dinucleotide phosphate (NADP) dehydrogenase and separated on this criterion from groups II and III, which have the activity. Group II species possess arogenate dehydrogenase enzyme (reactive with either NAD or NADP) sensitive to feedback inhibition by tyrosine, thereby separating from group III species whose corresponding enzyme was totally insensitive to feedback inhibition. The presence of prephenate/NADP dehydrogenase in group IV defined its separation from groups I and V, which lack this enzyme activity. Group I species possess an arogenate/NAD dehydrogenase that was highly sensitive to inhibition by tyrosine and a prephenate/NAD dehydrogenase of relative insensitivity to tyrosine inhibition. The opposite pattern of sensitivity/insensitivity was seen in group V species. These dehydrogenase characterizations are highly reliable for the keying of a given species to one of the five rRNA homology groups. If necessary, other confirmatory assays can be included using other aromatic pathway enzymes. These results further document the validity and utility of the approach of comparative enzymology and allostery for classification of microorganisms.

Alcaligenes↗
PubMed2006

C-reactive protein alters antioxidant defenses and promotes apoptosis in endothelial progenitor cells.

OBJECTIVE: C-reactive protein (CRP) has been suggested to participate in the development of atherosclerosis, in part, by promoting endothelial dysfunction and impairing endothelial progenitor cell (EPC) survival and differentiation. In the present study, we evaluated the effects of CRP on antioxidative enzymes, reactive oxygen species production, telomerase activity, and apoptosis in human circulating EPCs. METHODS AND RESULTS: EPCs, isolated from peripheral venous blood, were cultured in the absence or presence of native pentameric azide and lipopolysaccharide (LPS)-free CRP (0, 5, 15, and 20 microg/mL), N-acetylcysteine (NAC), hydrogen peroxide (H2O2) or monoclonal anti-CRP antibodies. Fluorescence-activated cell sorter (FACS) analysis was used for the measurement of intracellular H2O2 and superoxide (O2(-)) by loading cells with 2',7'-dichlorodihydrofluorescein diacetate (H2DCF-DA). Apoptosis was evaluated with Annexin V immunostaining and cytosolic cytochrome c expression. Western blot analysis was used for the determination of manganese superoxide dismutase (MnSOD) and glutathione peroxidase expression, and polymerase chain reaction enzyme-linked immunosorbent assay was used to assess telomerase activity. Incubation of EPCs with CRP caused a concentration dependent increase in reactive oxygen species (ROS) production and apoptosis, with an effect quantitatively similar to H2O2. This effect was attenuated during coincubation with NAC or anti-CRP antibodies. Furthermore, CRP altered EPC antioxidative enzyme levels, demonstrating a reduced expression of glutathione peroxidase and a significant increase in MnSOD expression. Transfection of EPCs with MnSOD-RNAi resulted in a reduction in CRP-induced ROS production, apoptosis, and telomerase inactivation. CONCLUSIONS: CRP, at concentrations known to predict cardiovascular events, may serve to impair EPC antioxidant defenses, and promote EPC sensitivity toward oxidant-mediated apoptosis and telomerase inactivation. These data further support a direct role of CRP in the development and/or progression of atherothrombosis.

Acetylcysteine↗
PubMed1991

Therapy of organophosphate poisoning in the rat by direct effects of oximes unrelated to ChE reactivation.

Isolated rat diaphragm preparations treated with soman or with the irreversible and oxime resistant cholinesterase (ChE) inhibitor S27 showed a considerable recovery of neuromuscular transmission (NMT) during incubation with the (bis)pyridinium oximes HI-6, HGG-12, P2S and obidoxime. In the soman-treated preparations this NMT recovery was predominantly caused by reactivation of acetylcholinesterase (AChE) but in the S27-treated preparations it was caused by a direct (pharmacological) effect unrelated to enzyme reactivation. Atropinized rats were artificially ventilated after injection with 3 x LD50 soman for 3 h and then treated with HI-6, i.e. at a time when oxime reactivation of soman inhibited ChE is no longer possible. Nevertheless, these rats started to breathe spontaneously and 50-60% survived more than 24 h, whereas all control animals (saline instead of HI-6) died within 10 min after artificial ventilation was terminated. In such animals no significant reactivation of ChE activity at various time intervals following HI-6 treatment was found, either in the diaphragms or in the brains. There was a significant amount of NMT (50%) in vitro in diaphragms obtained from these animals. This NMT did not improve in vitro in the presence of HI-6 and was not inhibited by soman administered to the medium. It is concluded that in this case the NMT found was based on synaptic adaptation to the continued inhibition of ChE and that the survival of the animals might be due to a combination of this synaptic adaptation and central direct effects of HI-6.

Animals↗
PubMed1994

Characterization and activation of naturally occurring abortive complexes of UDP-galactose 4-epimerase from Escherichia coli.

UDP-galactose 4-epimerase catalyzes the interconversion of UDP-galactose and UDP-glucose. The enzyme from Escherichia coli is a dimeric protein with an overall molecular weight of 79,000 that contains NAD+ very tightly but noncovalently bound in the enzymatic active site. NAD+ is the coenzyme for epimerization and is transiently reduced to NADH in the course of catalysis. All samples of highly purified UDP-galactose 4-epimerase contain significant amounts of NADH, and that purified after overexpression in E. coli cells contains a substantial amount of NADH. To the degree that NADH replaces enzyme bound NAD+ in the coenzyme binding site, the epimerase activity is decreased. The extinction coefficient at 345 nm for NADH in its binding site is estimated to be 3.3 mM-1 cm-1. 31P NMR spectroscopic and enzymatic analyses reveal that UDP-glucose, UDP-galactose, UDP, and UMP are gradually released from the purified enzyme upon addition of UMP or P1-5'-uridine-P2-methyl diphosphate (MeUDP). It is concluded that NADH associated with the purified enzyme is a component of inactive, abortive complexes (E-NADH-uridine nucleotide) that contain tightly bound uridine nucleotides in place of the epimerization intermediate UDP-4-keto-alpha-D-hexoglucopyranose. These complexes are produced in vivo in the course of bacterial growth. The enzymatic activity of purified epimerase is increased by reaction with 1,2-naphthoquinone-4-sulfonate, which oxidizes the NADH to NAD+. Compositionally defined abortive complexes (E-NADH-uridine nucleotide) containing UMP, UDP, or UDP-hexoses (Glc/Gal) have been prepared in vitro and subjected to activation by 1,2-naphthoquinone-4-sulfonate. All are activated at rates comparable to that for the purified enzyme, although those containing UMP and UDP-hexose are more readily activated than those containing UDP. The activity of the reactivated enzyme approaches that of the most highly active epimerase that has been reported from E. coli.

Carbohydrate Epimerases↗
PubMed1990

Muscle fiber typing in routinely processed skeletal muscle with monoclonal antibodies.

Muscle fiber typing is conventionally performed using mATPase enzyme histochemistry on cryostat sections. After pre-incubation of sections at pH 4.3, 4.6 and 10.3, based on the pattern of enzyme reactivity, the fibers can be classified in types I, II (subtypes A, AB and B) and the intermediate C (I and II) fibers. We have attempted to perform fiber typing of human psoas muscle by immunohistochemistry, using monoclonal antibodies R11D10 (specific for cardiac and type I skeletal myosin) and MY-32 (specific for fast muscle fibers) on cryostat as well as on paraffin sections. Staining of consecutive cryostat sections showed that type I fibers are R11D10 reactive whereas type II fibers are MY-32 reactive. Subtyping of type II fibers could not be performed by immunohistochemistry. Quantitative analysis of type I and II fibers showed that enzyme histochemical and immunohistochemical analysis are in close agreement.

Antibodies, Monoclonal↗
PubMed1988

Heating reactivation of rats aminolevulinic acid dehydratase in lead poisoning.

Heat reactivation (60 degrees C for 5 minutes) of the red blood cells aminolevulinic acid dehydratase activity was studied in lead treated rats (0.25 to 5.0 mg/kg b.w., i.p., for 4 weeks). Complete enzyme reactivation occurs with lead blood concentration up to about 70 mcg/dl. At higher blood lead concentrations, only a part of the enzyme can be restored by heating. The different mechanisms of aminolevulinic acid dehydratase activity inhibition by lead are discussed.

Animals↗
PubMed1988

Molecular defects in cytochrome oxidase in mitochondrial diseases.

Defects of cytochrome c oxidase (COX) show remarkable clinical, biochemical, and genetic heterogeneity. Clinically, there are two main groups of disorders, one dominated by muscle involvement, the other by brain dysfunction. Biochemically, the enzyme defect may be confined to one or a few tissues (reflecting the existence of tissue-specific isozymes) or affect all tissues. Immunologically reactive enzyme protein is decreased in some forms of COX deficiency but not in others. Because COX is encoded both by nuclear and by mitochondrial genes, COX deficiencies may be due to mutations of either genome and may offer useful models to study the communication between nuclei and mitochondria. We have isolated full-length cDNA clones encoding human COX subunits IV, Vb, and VIII and a partial-length clone for subunit Va. These clones are being used as probes to analyze the DNA and RNA of patients with COX deficiency.

Brain Diseases↗
PubMed2004

Effect of hypergravity on carboanhydrase reactivity in inner ear ionocytes of developing cichlid fish.

It has been shown earlier that hypergravity slows down inner ear otolith growth in developing fish. Otolith growth in terms of mineralization mainly depends on the enzyme carboanhydrase (CA), which is responsible for the provision of the pH-value necessary for calcium carbonate deposition. Larval siblings of cichlid fish (Oreochromis mossambicus) were subjected to hypergravity (3 g, hg; 6 h) during development and separated into normally and kinetotically swimming individuals following the transfer to 1 g (i.e., stopping the centrifuge; kinetotically behaving fish performed spinning movements). Subsequently, CA was histochemically demonstrated in inner ear ionocytes (cells involved in the endolymphatic ion exchange) and enzyme reactivity was determined densitometrically. It was found that both the total macular CA-reactivity as well as the difference in reactivities between the left and the right maculae (asymmetry) were significantly lower (1) in experimental animals as compared to the 1 g controls and (2) in normally swimming hg-animals as compared to the kinetotically behaving hg-fish. The results are in complete agreement with earlier studies, according to which hypergravity induces a decrease of otolith growth and the otolithic calcium incorporation (visualized using the calcium-tracer alizarin complexone) of kinetotically swimming hg-fish was higher as compared to normally behaving hyper-g animals. The present study thus strongly supports the concept that a regulatory mechanism, which adjusts otolith size and asymmetry as well as otolithic calcium carbonate incorporation towards the gravity vector, acts via activation/deactivation of macular CA.

Animals↗
PubMed1978

Comparative immunological studies on arylsulfatase in bacteria of the family Enterobacteriaceae: occurrence of latent arylsulfatase protein regulated by sulfur compounds and tyramine.

The arylsulfatases of 21 strains of the family Enterobacteriaceae were compared by measuring their enzymatic activities and immunological reactivities. Enzyme formation under repressing, nonrepressing, and derepressing conditions was tested. Antiserum prepared against pure arylsulfatase from Klebsiella aerobgenes W70 was tested against the enzyme extracts from the strains using double diffusion, quantitative precipitation, and immunoelectrophoresis. No close relationship was found between arylsulfatase activity and immunological cross-reactionship was found between arylsulfatase activity and immunological cross-reactivity. The strains in the family Enterobacteriaceae could be divided into two groups on the basis of the immunological properties of their enzyme. Antisera formed a precipitin band with both active and inactive enzyme proteins from Escherichia, Citrobacter, Salmonella, Klebsiella, and Enterobacter, but not with the proteins from Serratia, Proteus, and Erwinia, even though some strains of these species had enzyme activity. It was also found that the formation of arylsulfatase proteins, irrespective of whether they had enzyme activity, were under regulation by sulfur compounds and tyramine.

Arylsulfatases↗
PubMed1978

Absence of evidence for an intermediate in the deacetylation of acetylchymotrypsin.

A recent paper [Chibber, B. A. K., Tomich, J. M., Mertz, E. T. & Viswanatha, T. (1977) Proc. Natl. Acad. Sci. USA 74, 510-514] presented evidence that was taken to support the existence of an intermediate in the deacetylation of acetylchymotrypsin. It was observed that deacylation, as measured by following the decrease in [(14)C]acetylchymotrypsin (decrease in acid-precipitable radioactivity), occurred at 1/10 the rate of reactivation, as measured by return of activity toward N-acetyl-L-tyrosine ethyl ester. Our experiments have shown that, at pH 6, the deacylation rate constant (measured by the loss of [(14)C]acetylchymotrypsin and by the formation of [(14)C]acetate) is identical (within experimental error) with the rate constant for reactivation (measured by determining the activity of aliquots of reactivating enzyme against N-acetyl-L-tryptophan ethyl ester) and with K(cat) for the turnover of p-nitrophenyl acetate by alpha-chymotrypsin. Part of the 10-fold greater reactivation rate observed by Chibber et al. has been shown to be due to the presence of 10% (vol/vol) isopropanol in their reactivation mixture, and it is argued that the balance of the effect is a manifestation of the "indole effect" produced by the simultaneous presence of 10 mM N-acetyl-L-tyrosine ethyl ester throughout the reactivation experiments. The results presented are entirely consistent with the three-step mechanism of catalysis by alpha-chymotrypsin and negate the existence of the proposed additional acetyl-enzyme intermediate.

Acetylation↗