Partially carboxylated prothrombins. I. Comparison of activation properties and purification of 1- and 0-carboxyglutamyl variants.

In addition to the 7-, 5- and 2-carboxyglutamyl varieties of dicoumarol-induced prothrombins (Malhotra, O.P. (1979) Thromb. Res. 15, 427-463), we have isolated two more atypical prothrombins, one containing 1.1 +/- 0.1 gamma-carboxyglutamic acid, '1-carboxyglutamyl prothrombin,' and the other less than 0.2, '0-carboxyglutamyl prothrombin.' Both variants showed a single component by analytical polyacrylamide-gel electrophoresis in the absence and in the presence of sodium dodecyl sulfate and contained antigenic activity indistinguishable from that of normal prothrombin. The pI of both proteins as assessed by electrofocusing was 4.835 +/- 0.015, compared with 4.58 for 10- and 7-, 4.75 for 5- and 4.81 for 2-carboxyglutamyl materials. By the two-stage prothrombin assay procedure, the 1- and 0-carboxyglutamyl variants generated thrombin, respectively 19 and 13% of normal prothrombin, and their activation times ranged from 4 to 7 h, compared with 7 min for normal. Kinetic studies, utilizing the one-stage coagulation assay, showed that both Km and tmin (minimal clotting time) increase proportionally with the decrease of gamma-carboxyglutamyl residues (from 10 to 7, 5, 2, 1 and 0 gamma-carboxyglutamic acids). Each of the five (partially) acarboxy prothrombins owe their clotting activity to gamma-carboxyglutamyl residues and not to the presence of some normal prothrombin molecules.

Partially carboxylated prothrombins. II. Effect of gamma-carboxyglutamyl residues on the properties of prothrombin fragment 1.

Purified prothrombin fragments 1 derived from normal (10-carboxyglutamyl) and dicoumarol-induced 7-, 5-, 2-, 1-, and 0-carboxyglutamyl prothrombins contained the same number of gamma-carboxyglutamyl residues as their respective parent molecules. The effect of gamma-carboxyglutamyl residues was more pronounced on the fragments 1 than on the prothrombins. Consequently, the pI values of the fragments 1 were very well differentiated, with normal fragment 1 focusing at pH 3.58, 7-carboxyglutamyl fragment 1 at 3.79, 5- at 3.97, and 2- at pH 4.29. Similarly, by agar gel electrophoresis, normal fragment 1 was the most mobile, followed by 7-, 5-, 2-, 1- and lastly 0-carboxyglutamyl fragment 1. Because of Ca2+ being bound to the carboxyglutamyl residues, the electrophoretic mobility of normal fragment 1, in the presence of Ca2+, was reduced the most, followed by 7-, 5- and then 2-carboxyglutamyl fragment 1, while the mobilities of the 1- and 0-carboxyglutamyl fragments 1 were not affected. In contrast to their parent molecules, all of the fragments 1 in the presence of EDTA gave negative immunoprecipitation reactions against antibodies produced against normal prothrombin. In the presence of Ca2+, conversely, the fragments 1 containing comparable amounts of antigenic activity all gave positive reactions. However, the intensity of the immunoprecipitates varied, as normal fragment 1 gave the most prominent immunoprecipitation reaction, consecutively followed by 7-, 5-, 2-, 1- and lastly 0-carboxyglutamyl fragment 1 where the precipitation was so faint that it was hardly visible.

Kinetics of inactivation and molecular asymmetry of NAD-specific glyceraldehyde-3-phosphate dehydrogenase of Pisum sativum.

Glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate: NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12) has been purified to homogeneity from pea seeds. The purified enzyme gave a single protein band on polyacrylamide gel electrophoresis (with and without sodium dodecyl sulfate; subunit molecular weight 38 000). It is free from bound nucleotides. The kinetics of heat inactivation of the crude enzyme extract as well as the purified enzyme are biphasic, in that exactly half of the activity is destroyed more rapidly than the residual half. The data are consistent with the rate equation: (formula: (see text): where A0 and A are activities at times zero and t, respectively, and k1 and k2 are first-order rate constants for the fast and slow phases, respectively. Addition of NAD+ slows down thermal inactivation, without altering the overall kinetic pattern. The activity lost due to the fast component (k1) of the reaction is regained on colling ('annealing'), whereas the slow reaction (k2) is not reversed, suggesting the following scheme: formula (see text): This is confirmed by plotting the activity after 'annealing' against initial period of heating. A single first-order rate constant (k2) is observed. The enzyme possesses about one reactive SH group per subunit which can be titrated with 5,5'-dithio-bis-(2-nitrobenzoic acid). Blocking of these groups inactivates the enzyme. Inactivation with 20 micrometer N-ethylmaleimide and 30 micrometer iodoacetate (at pH 8.6 and 33 degrees C) follows simple first-order kinetics (rate constants 0.099 and 0.139 min-1, respectively), suggesting that all SH groups react equally readily with these reagents. Reaction of the enzyme with 0.6 micrometer p-chloromercuri benzoate, however, shows biphasic kinetics similar to thermal inactivation. The reaction of p-chloromercuri benzoate with partially heat-inactivated enzyme (residual activity 37.5%) follows simple first-order kinetics. The molecular asymmetry demonstrated by these results must arise from the unique quaternary structure of the enzyme molecule, which is apparently made up of chemically identical subunits (pseudo-isologous association).

Structural and functional consequences of subunit interactions in glyceraldehyde 3-phosphate dehydrogenase.

A variety of species of GPDH undergo acylation at two of the four active cystein sites per mole of tetrameric enzyme. This reaction requires tightly bound NAD+, a situation restricted to two of the four NAD sites per tetramer. S leads to N acyl transfer from cysteins to lysine in the diacyl enzyme yields an inactive enzyme. The thiol ester bond of acyl enzyme is activated by NAD+ and NADH for the group transfer and reduction reactions, respectively. In furyl acryloyl-GPDH this activation is accompanied by large acyl-spectral shifts, a "blue shift" with NADH and a "red shift" with NAD+. The group transfer reaction as well as spectral shifts show biphasic kinetics. The amplitude of the fast phase of NAD+-induced spectral change in apo-enzyme is equal to that of the fast phase in phosphorolysis (or arsenolysis) at low [NAD+]. The kinetic pattern of spectral shifts by NAD+ and NADH are complementary; the amplitude of the fast phase in one is equal to that of the slow phase in the other. It has been proposed that the acyl enzyme exists in two conformational states. The relative proportion of these states varies with the extent of covalent (acyl group) or non-covalent (NAD+ or NADH) ligation in a manner consistent with the allosteric model of Monod, Wyman and Changeux. These conclusions apply equally to the true substrate acyl enzyme. With 1,3-diphosphoglycerate, a tetra-acylated enzyme is obtained. Two of these four acyl groups react very much faster than the remaining two. A comparison of their specific rates with the steady state turnover numbers indicates that only the less reactive two acyl groups govern the turnover number of the enzyme.

Purification and characterization of dicoumarol-induced prothrombins. Evidence of 5-Gla variant with two (or more) polypeptide chains.

A 5-Gla prothrombin, adsorbable onto barium oxalate and containing an admixture of one- and two-chain molecules, has been purified from the plasma of steers on a dicoumarol regimen for three years. Double-chain molecules were not detected in any of our six other variants containing zero to nine Gla residues. In this double-chain variant, the peptide bond between Arg52-Asn appears to be missing, as the preparation showed amino-terminal alanine and aspartic acid, and released both double- and single-chain F1 upon digestion with thrombin. The molecular masses of the F1 and its heavy chain were 24,000 and 19,000 daltons, respectively. The double-chain variant was similar to its single-chain counterpart [Malhotra, O.P. (1979) Thromb. Res. 14, 439-448] in that it (a) yielded thrombin equivalent to 30% of normal in three hr, (b) moved in the alpha 1 region in the absence of calcium ions and between the alpha 1 and alpha 2 macroglobulins in the presence of calcium ions, (c) contained five gamma-carboxyglutamyl residues (Gla), and (d) showed comparable antigenic activity with normal prothrombin.

Electroimmunoassay of prothrombin.

Electroimmunoassay of normal (10-Gla) and Gla-deficient prothrombins containing 0 to 9 Gla (gamma-carboxyglutamyl) residues performed in EDTA against anti- (normal) prothrombin showed that each of the Gla-deficient proteins contained as much antigenic activity as does the normal molecule. In the presence of Ca2+, however, normal prothrombin appeared to show the least while 0- to 2-Gla variants, the most activity. This anomaly arises from the presence of antibodies (Ca-IIAb) which form antigen-antibody (Ag-Ab) complexes when the conformation of the antigen is stabilized in Ca2+. For example, upon fractionation of the antisera and mixing Ca-IIAb (bound to 10-Gla prothrombin, Affi-gel column and eluted by replacing Ca2+ with EDTA) with those reacting with prethrombin1 (non-Gla portion of the molecule), rocket heights in Ca2+ decreased the most with 10- and 9-Gla (32%), followed by 13% with 8-Gla, 2% with 7-Gla and none with 2-Gla prothrombins--indicating that Ca-IIAb do cross react with 7-, 8- and particularly 9-Gla prothrombins. This was also confirmed by the fact that anti 7-Gla but not anti 0-Gla sera contained some antibodies which reacted only in the presence of Ca2+.

Monoclonal antibodies to prothrombin.

Hybridoma technology was used for the production of murine monoclonal antibodies to bovine normal prothrombin. Hybrid cell cultures were assayed for the production of antibodies, both in the absence and presence of calcium ions, by Enzyme-Linked Immunosorbent Assay (ELISA). Antibody-producing cell lines were cloned two times and grown as ascites tumors. Monoclonal antibodies (McAb), isolated by affinity chromatography (Protein A-Sepharose), were tested for their affinity for normal (10-Gla) and dicoumarol-induced abnormal prothrombins containing 2, 5, 7, 8 and 9 gamma-carboxyglutamyl (Gla) residues. A total of 24 McAb were obtained and the immunoglobulins were of the IgG1 subclass. Nine of the twenty-four McAb did not require Ca2+ for the formation of Ag-Ab complexes, and reacted equally with normal and Gla-deficient prothrombins. These antibodies had affinity for prethrombin1 (P1) but not for the Gla-containing prothrombin fragment1 (F1) portion of the molecule. In contrast, the 15 Ca2+-dependent McAb reacted with F1 but not with P1. They discriminated the abnormal prothrombins based upon their Gla content. For example, though all the Ca2+-dependent McAb formed Ag-Ab complexes with 9-, essentially none formed with 5- or less-Gla prothrombins. [Some reacted equally with 9- and 10-Gla (normal) prothrombin while others had only 25% of normal affinity for 9-Gla isomer]. Only four and twelve of the 15 McAb had some affinity for 7- and 8-Gla variants, respectively. These results show that antibodies which react with the Ca2+-stabilized conformation of prothrombin are not specific for normal prothrombin, as has been reported in the literature.

Application of short column gel permeation in the study of protein-protein interactions.

A simple and rapid procedure based on the gel filtration principle is described together with its applicability to the study of protein-protein interactions including subunit-subunit and enzyme-enzyme interactions. Using this procedure, it is shown that phosphoglycerate kinase (PGK) and glyceraldehyde-3-phosphate dehydrogenase (GPDH) interact with a stoichiometry of one PGK molecule combining with one monomeric subunit of GPDH. This interaction has been observed with both enzymes being from the same, as well as from different, species. The Kd values for rabbit muscle PGK and porcine muscle GPDH complex and that for the rabbit muscle PGK and yeast GPDH complex are found to be (4.5 +/- 2.0) x 10(-7) M and (6.5 +/- 1.7) x 10(-7) M, respectively. The specificity of bienzyme association is stronger when enzymes are from the same species than when they are from different species.

Critical illness polyneuropathy: how often do we diagnose it?

Neuromuscular weakness in critically ill patients is a diagnostic challenge. Critical illness polyneuropathy, an important cause of failure to wean from assisted ventilation is often missed due to lack of suspicion and initiative to undertake regular bedside neurological and electrophysiological examinations in critically ill patients. We report two cases who developed motor weakness while receiving mechanical ventilation in whom axonal neuropathy was diagnosed on electrophysiological studies, establishing a diagnosis of critical illness polyneuropathy. Both patients had evidence of sepsis and multiorgan failure. One case could be successfully weaned off and weakness improved while other succumbed to the underlying illness.

Pulmonary functions in patients with epidemic dropsy.

Pulmonary functions were performed in thirteen patients with epidemic dropsy. The epidemic occurred in Delhi in 1998 during which 102 patients with epidemic dropsy reported to our medical unit. Other investigations included chest radiograph, ECG, liver and renal function tests. There was a restrictive ventilatory defect with diminution of diffusion capacity for carbon monoxide in these patients. Echocardiogram was done in seven of these patients and was normal. The cause of breathlessness and restrictive ventilatory defect is likely to be non-cardiogenic pulmonary oedema.

Primary pulmonary lymphoma presenting as lung mass.

The commonest cause of lung mass in an elderly patient is bronchogenic carcinoma. We are reporting an unusual case of lung mass that was diagnosed following exploratory thoracotomy and pneumonectomy. Sputum examination, bronchoscopy and percutaneous fine needle aspiration cytology were inconclusive. On histopathology, a diagnosis of non-Hodgkin's lymphoma (NHL) was made. There was no involvement of any other site on detailed work up. The patient was advised chemotherapy.

A clinico-haematologic profile of paroxysmal nocturnal haemoglobinuria.

Clinico-haematological parameters in sixteen patients of paroxysmal nocturnal haemoglobinuria (PNH) are presented. Their modes of presentation included recurrent episodes of cola-coloured urine (6/16), refractory anaemia (9/16) and predominant thrombotic manifestations (1/16). Laboratory investigations revealed the presence of anaemia (16/16), reticulocytosis (14/16), thrombocytopenia (11/16), leucopenia (5/16) and cellular bone marrow (14/16). Two patients had hypoplastic bone marrow initially but subsequently developed PNH. The patients were treated with haematinics, prednisolone (16/16) and oxymethalone (2). Prednisone was effective in suppressing haemolytic episodes. Oxymethalone given to the 2 patients with hypoplastic bone marrow resulted in amelioration of anaemia in one but no effect in the other patient.

Pregnancy with idiopathic thrombocytopenic purpura.

The management of ITP in pregnancy remains controversial, particularly with reference to labour management. Thirteen pregnancies in 9 women with ITP are analysed with respect to maternal and neonatal outcome. One pregnancy culminated in spontaneous abortion. Ten infants were born by vaginal delivery and two by Caesarean section. There were no maternal or perinatal deaths. Maternal morbidity was not increased significantly due to ITP and none of the infants had purpuric manifestations even with low platelet counts. It is concluded that the obstetric management of these patients should be individualised and should not be based on platelet count alone.

Differential binding of NAD+ to acyl glyceraldehyde-3-phosphate dehydrogenase and its role in the acyl group transfer reaction.

Enzyme protein fluorescence of di-furylacryloyl-glyceraldehyde-3-phosphate dehydrogenase (di-FA-GPDH:lambda max.excitation 290 nm, lambda max.emission 338 nm) is quenched about 28% on saturation with NAD+. Results of fluorometric titration of di-FA-GPDH with NAD+ suggest the presence of two tight and two loose coenzyme binding sites (Kdiss. 0.1 and 6.0 microM, respectively). Initial rates of the NAD(+)-dependent reaction of di-FA-GPDH with arsenate and phosphate and of mono-FA-GPDH with phosphate have been determined at varying coenzyme concentrations. The data suggest that binding of NAD+ at the tight sites does not activate the acyl group for its reaction with the acceptor (phosphate or arsenate). The group transfer reaction is dependent only on NAD+ binding to the loose sites, which carry the acyl group. The large difference in the NAD+ binding affinity to the two types of sites and their different effects on the group transfer reaction impart a sigmoidal shape to the rate versus [NAD+] plots. The sigmoidicity is abolished if the reactive SH groups at the unacylated sites are blocked by carboxymethylation.

Site-site heterogeneity in isocitrate lyase of castor seed endosperm: evidence from kinetics of inactivation by tetranitromethane.

Inactivation of isocitrate lyase (native and EDTA-dialysed) by excess tetranitromethane (TNM) exhibits, biphasic kinetics, in which half of the initial activity is lost in a fast and the remaining half in a slow phase each following the pseudo-first order kinetics. Rate constants of the two phases are proportional to the TNM concentration. High succinate concentration protects the enzyme against TNM inactivation only in the slow phase without any effect on the fast phase. With the EDTA-dialysed enzyme, no such protection (against inactivation by TNM) is observed in the presence of succinate or Mg2+ ions. Addition of both these ligands together brings about protection against the slow phase (as with the native enzyme). It has been proposed that the site-site heterogeneity of isocitrate lyase is a consequence of its quaternary structure constraints.

Kinetics of thermal inactivation and quaternary structure symmetry of rabbit muscle glyceraldehyde 3-phosphate dehydrogenase.

Kinetics of thermal inactivation of apo-glyceraldehyde 3-phosphate dehydrogenase have been investigated under various conditions. At most pH values, the loss of enzyme activity takes place in two phases, a fast and a slow phase. The data correspond to the rate equation A = Afast.e-kfast.t + Aslow.e-kslow, where A is the observed residual activity (expressed as % of initial activity) at time t, Afast and Aslow are amplitudes (expressed as % of initial activity, so that Afast + Aslow = 100) and kfast and kslow the rate constants of the fast and slow phases, respectively. At pH 9 or below, Afast = Aslow = 50%. As pH is increased above 9, Afast increases gradually till at pH 10 or above when it accounts for the entire initial activity (single exponential decay). The rate constants of the two phases are strongly affected by the nature of the buffer, temperature and pH, but the amplitudes depend on pH alone. It has been suggested that the tetrameric enzyme exists in two conformations of different molecular symmetry, namely C2 (two pairs of sites of unequal stability, predominating at pH 9 or below) and D2 symmetry (four equivalent sites, predominating at pH 10 or above). The C2 in equilibrium D2 transformation is found to be highly cooperative with midpoint at pH 9.6.

Effect of substrates and coenzymes on the molecular symmetry-related properties of horse liver and yeast alcohol dehydrogenases.

Thermal inactivation of horse liver alcohol dehydrogenase (LADH) exhibits the following biphasic kinetics A = Afast.e-Kfast.t + Aslow.e-Kslow.t Where A is the per cent residual activity at time t,Afast and Aslow are amplitudes (expressed as % of initial activity) and kfast and kslow first-order rate constants of the fast and slow phases, respectively. For apoenzyme, Afast = Aslow = 50% of the initial activity under all conditions of temperature and pH. On the addition of a substrate or coenzyme ligand, there is a ligand concentration-dependent increase in per cent Aslow and a decrease in kslow. At sufficiently high ligand concentration, the entire time-course of inactivation can be described as a single exponential decay. The variations of per cent Aslow and of kslow with ligand concentration are consistent with the existence of two binding sites of different ligand affinities. Inactivation of LADH by excess EDTA also exhibits a similar biphasic kinetics with Afast = Aslow = 50% of the initial activity. Addition of ethanol or NAD+ brings about a concentration-dependent decrease in kfast and kslow without affecting amplitudes of the two phases. The NAD+ concentration-dependence of this decrease is consistent with a single dissociation constant for the coenzyme. Inactivation of yeast alcohol dehydrogenase by heat or excess EDTA can be represented as a single exponential decay of activity under all conditions of temperature and pH in the absence as well as presence of ethanol or NAD+. Implications of these results for the molecular symmetry of the two oligomeric enzymes in solution are discussed.

Steady-state kinetic properties of phosphoglycerate kinase of mung beans.

Steady-state kinetics of the action of mung bean phosphoglycerate kinase have been investigated using 3-phosphoglycerate and ATP as substrates in the presence of Mg2+ ions. Keeping a constant and high Mg2+ concentration and varying the concentration of one of the substrates (ATP or 3-phosphoglycerates) at several fixed concentrations of the other substrate (3-phosphoglycerate or ATP), the Km values of Mg.ATP2- and 3-phosphoglycerate were found to be 0.42 and 0.60 mM, respectively. These values are independent of the concentration of the other substrate. A limiting value of Vmax, where the enzyme is saturated with both the substrates, was found to be 39.4 mumoles product formed per min per mg enzyme protein. This corresponds to a turnover number equal to 31.5 sec-1 (for molecular weight of the enzyme equal to 48,000). If [Mg2+] and [ATP4-] are held equal and varied together at several fixed concentrations of 3-phosphoglycerate, deviations from Michaelis-Menten kinetics (non-linear Lineweaver-Burk plots) are observed at lower values of ATP4- and Mg2+ (less than 0.1 mM), giving rise to apparent sigmoidicity in the rate versus [ATP4-] plots. It has been suggested that the real substrate for this enzyme is the Mg.ATP2- complex (and not free ATP4-). The complex dissociates at lower values of [Mg2+] and [ATP4-]. The resulting disproportionate decrease in the concentration of the complex brings about a steeper fall in the rate of reaction than is required by the Michaelis-Menten equation, giving rise to an apparent sigmoidicity.(ABSTRACT TRUNCATED AT 250 WORDS)

Phosphoglycerate kinase--glyceraldehyde-3-phosphate dehydrogenase interaction: reaction rate studies.

Rate studies using phosphoglycerate kinase (PGK)--glyceraldehyde-3-phosphate dehydrogenase (GPDH) enzyme pair have been carried out to distinguish between the two mechanisms of intermediate metabolite transfer, namely diffusion through the solvent versus "substrate channelling" within an enzyme-enzyme complex. A procedure has been described for the assay of the rates of PGK-catalysed and the PGK-GPDH coupled reactions at high (saturating) GPDH concentration. With PGKs of rabbit muscle and yeast, the coupled reaction proceeded faster than the PGK-catalysed reaction. At a high salt concentration (0.5 M KCl), where a PGK-GPDH complex is known to dissociate, the two reactions proceeded at almost equal rates. At fixed PGK concentration, the rate of the coupled reaction at high (saturating) GPDH concentration varied with the nature (biological origin) of the latter enzyme. In the presence of 0.5 M KCl, the saturating rate values with different GPDHs were almost equal. The PGK-catalysed reaction exhibited typical Michaelian behaviour on varying the substrate concentrations (linear double reciprocal plots). The Km values for 3-PGA (0.51 mM) and ATP (0.40 mM) were independent of the concentration of the second substrate. The double reciprocal plots for the coupled reaction showed downward curvature, i.e. activation at higher substrate concentrations. The ratio of the rate of the coupled reaction: the rate of the PGK catalysed reaction was found to be a function of the nature of PGK, nature of GPDH, nature of buffer, pH, salt concentration and substrate concentrations. The ratio varied between close to unity at low substrate concentrations, to three when the Vmax values of the two reactions were compared. At low substrate concentrations, the rate of the coupled reaction became independent of the nature of GPDH. It has been suggested that in the PGK-GPDH pair, the intermediate metabolite (BPG) is transferred directly from one enzyme to the other within an enzyme-enzyme complex, except at high salt or low substrate concentrations. Under the latter conditions, data were consistent with metabolite transfer by diffusion. Implications of these results for coupled enzyme assays have been discussed.

Regulatory properties and active site groups of cytosolic mung bean pyruvate kinase.

Properties of mung bean pyruvate kinase were studied and the active site groups were derived. Metabolites like AMP, glucose, glucose-6-phosphate, fructose-6-phosphate, fructose-1, 6-bisphosphate, 3-phospho-glycerate, isocitrate, malate and alpha-ketoglutarate had practically no effect on pyruvate kinase activity. Alanine, serine, glutamine, methionine and GMP had a weak activating effect on the enzyme. Some metabolites such as ATP, GTP, and UMP were found to be weakly inhibitory. Moderate to strong inhibition was observed with citrate, succinate, glutamate and oxalate. Inhibition brought about by ATP and citrate when present together showed synergistic effect. Inhibition by citrate was non-competitive with respect to both PEP and ADP suggesting the presence of a regulatory site. Mung bean pyruvate kinase showed half optimal activity at pH 6.6 and 8.9 at saturating concentrations of PEP, ADP and Mg2+. Small concentrations of the SH specific reagents, namely iodoacetamide (0.1 and 0.2 mM), N-ethylmaleimide(0.05-0.1 mM) and p-chloromercuribenzoate (0.1 mM) inactivated the enzyme; single exponential loss of activity was observed in each case. Photooxidation of the enzyme in the presence of methylene blue (100 and 200 micrograms/ml) and rose bengal (5 and 10 micrograms/ml) also led to a single exponential activity decay. When the enzyme was treated with diethyl pyrocarbonate (DEP), a time dependent exponential decay in its activity was observed with a parallel increase in absorbance at 240 nm. PEP protected the enzyme against inactivation by DEP. Reagents specific for tyrosine (iodine and tetranitromethane) and tryptophan residues (N-bromosuccinimide) residues had no effect. These observations confirm that SH and imidazole groups are vital for the activity of the enzyme.