Alterations in trehalase solubility during development in the cellular slime mould Dictyostelium discoideum

Previous studies have indicated that during development in the slime mould Dictyostelium discoideum, compartmentation of the isoenzymes of trehalase (alpha, alpha'-trehalose 1-D-glucohydrolase, (EC 3.2.1.28) occurs between the extracellular and intracellular environments. The compartmentation of trehalase between soluble and particulate cell fractions was examined in this work. The trehalase present in crude homogenates prepared during the first 12 h of development was completely soluble. Starting at about the pseudoplasmodial stage (i.e. the 14th hour of development), trehalase activity became associated with insoluble cellular material and this increased to a maximal value in homogenates from mature sorocarps, where 50% of the activity was insoluble. Spore cells accounted for only 2 to 3% of the trehalase associated with mature sorocarps, with the remaining 97% being localized in stalk cell material. Although trehalase recovered from spores was completely soluble, more than half of that from the stalk was recovered in the buffer-insoluble pellet fraction.     

Purification and characterization of a novel mycolic acid exchange enzyme from Mycobacterium smegmatis

We have isolated and purified to homogeneity an alpha,alpha'-trehalose 6-monomycolate:alpha,alpha'-trehalose mycolyltransferase (trehalose mycolyltransferase) from Mycobacterium smegmatis that catalyzes the exchange of a mycolyl group between trehalose, trehalose 6-monomycolate (TM), and trehalose 6,6'-dimycolate (TD). This enzyme was prominent in M. smegmatis and it catalyzed the following reactions. TM + [14C]trehalose in equilibrium [14C]TM + trehalose [14C]TM + TM in equilibrium [14C]TD + trehalose This enzyme was purified by (i) ammonium sulfate fractionation, (ii) QAE-Sephadex A-50 column chromatography, (iii) gel filtration on a Sephadex G-75 column, and (iv) SP-Sephadex C-50 column chromatography. The purified protein yielded a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and its molecular weight was estimated to be 25,000. This enzyme was a glycoprotein, had no cofactor requirement, and was highly specific for alpha,alpha'-trehalose as the mycolate acceptor. It was less specific for the acyl donor group since the palmitoyl group in trehalose 6-monopalmitate was easily exchangeable. There was no TM acylhydrolase activity in the purified enzyme, suggesting that it is probably associated with the anabolic pathway of mycolic acid metabolism. We postulate the formation of a mycolyl-enzyme intermediate in this reaction. Such an intermediate could play a central role in the transfer of mycolic acid to form the prominent cell wall components of mycobacterial TD and possibly murein-arabinogalactan-mycolate.     

Intracellular trehalase of a hybrid yeast

1. The trehalase found in an extract prepared from a yeast strain that cannot ferment trehalose was studied and characterized. The enzyme is highly specific for trehalose with K(m) 1.02x10(-2)m, and an optimum pH of 6.9. 2. It is inhibited by glucose and by trehalose 6-phosphate, and does not facilitate any significant transglucosylations. 3. pK values 7.7 and 5.8 were detected for the groups associated with binding of the non-ionized substrate to the enzyme. 4. The trehalase was found to be highly labile and was inhibited by thiol-binding reagents. 5. The possible role of this enzyme in the trehalose-dissimilation patterns in the yeast cell was evaluated.     

Intestinal and renal origin of trehalase activity in rabbit amniotic fluid

To utilize specific fetal markers in amniotic fluid for prenatal detection of fetal anomalies, it is necessary to determine the precise tissue origin of these markers. In rabbit fetuses, we distinguished between intestinal and renal forms of trehalase (alpha,alpha'-trehalose-1-D-glucohydrolase, EC 3.2.1.28) in amniotic fluid on the basis of differences in net electric charges. Trehalase was solubilized from purified brush-border membranes of fetal rabbit kidney and intestine by Triton X-100 treatment, whereas the trehalase activity in amniotic fluid was soluble. The kinetic properties of trehalase from intestine, kidney and amniotic fluid were very similar. The Mr of the soluble amniotic fluid trehalase was between 72,600 and 66,300 from hydrodynamic parameters, depending on the amount of sugar bound to the enzyme, and 48,500 by radiation inactivation, a method which detects only the protein part of the enzyme. For membrane-bound trehalase from kidney and intestine in situ the radiation inactivation method also gave a molecular size of around 49,000. Isoelectric focusing of freshly solubilized membranes allowed us to distinguish between renal and intestinal forms of trehalase in rabbit fetuses on the basis of different isoelectric points. Each trehalase form was also present in the amniotic fluid but in varying proportions depending on the gestational age at which the amniotic fluid was collected. The results suggest that early in gestation amniotic fluid trehalase activity originates exclusively from the fetal kidney but that more and more intestinal enzyme is released into the amniotic cavity as the fetus develops. Similar results were also obtained when ion-exchange chromatography was used to separate the various trehalase forms. The development of trehalase activity in rabbit fetal kidney and intestine correlates well with its occurrence in the amniotic fluid; trehalase activity in the kidney develops early in gestation whereas the intestinal trehalase activity develops just before term.     

Purification and characterization of amphiphilic trehalase from rabbit small intestine

Rabbit intestinal trehalase (alpha,alpha-trehalose glucohydrolase, EC 3.2.1.28) was solubilized with Triton X-100 and purified in the presence of EDTA. The purified enzyme was homogeneous on polyacrylamide gel electrophoresis in the presence of Triton X-100 or SDS. It showed amphiphilic properties on gel filtration. polyacrylamide gel electrophoresis, charge-shift electrophoresis and phenyl-Sepharose chromatography. Its molecular weight was estimated to be about 330 000 by gel filtration under nondenaturing conditions and in the presence of Triton X-100, the value being in satisfactory agreement with the sum of the weight of one Triton X-100 micelle and twice the molecular weight (105 000) of purified hydrophilic trehalase which had been deprived of the anchor segment. The two purified trehalases gave almost the same molecular weights (about 75 000) on SDS-polyacrylamide gel electrophoresis. These results suggest that intestinal trehalase consists of two subunits with a molecular weight of 75 000 and that its anchor segment is small (less than 5000). Triton X-100 extracts freshly prepared from intestinal microvilli essentially showed one form of trehalase, which behaved on phenyl-Sepharose and Con A-Sepharose chromatography in the same manner as purified amphiphilic trehalase.     

Construction, assembling and application of a trehalase-GOD enzyme electrode system

Trehalose is a disaccharide important in foods, serving as a glucose source in many and also as an additive in the food preparation. Because of its peculiar physico-chemical properties it plays an important role as preservative in drying and deep-freezing treatments. A new biosensor for trehalose determination has been realized by means of a flow system, based on a reactor in which the trehalase enzyme catalyses its hydrolysis into two alpha,d-glucose molecules, and a GOD (glucose oxidase) amperometric biosensor is employed for the glucose determination. The optimum operative conditions have been laid out and a particular attention has been paid to the immobilization procedure of the two enzymes. The electrode used is of the SPE (screen-printed electrode) type and has been activated with the Prussian Blue (PB) and then assembled using GOD immobilized with Nafion. The reactor has been prepared with the trehalase enzyme chemically immobilized on an Immunodyne ABC membrane. As demonstration of its utility, the biosensor has been tested on a real sample of Boletus edulis mushroom.     

A novel trehalase from Mycobacterium smegmatis - purification, properties, requirements

Trehalose is a nonreducing disaccharide of glucose (alpha,alpha-1,1-glucosyl-glucose) that is essential for growth and survival of mycobacteria. These organisms have three different biosynthetic pathways to produce trehalose, and mutants devoid of all three pathways require exogenous trehalose in the medium in order to grow. Mycobacterium smegmatis and Mycobacterium tuberculosis also have a trehalase that may be important in controlling the levels of intracellular trehalose. In this study, we report on the purification and characterization of the trehalase from M. smegmatis, and its comparison to the trehalase from M. tuberculosis. Although these two enzymes have over 85% identity throughout their amino acid sequences, and both show an absolute requirement for inorganic phosphate for activity, the enzyme from M. smegmatis also requires Mg(2+) for activity, whereas the M. tuberculosis trehalase does not require Mg(2+). The requirement for phosphate is unusual among glycosyl hydrolases, but we could find no evidence for a phosphorolytic cleavage, or for any phosphorylated intermediates in the reaction. However, as inorganic phosphate appears to bind to, and also to greatly increase the heat stability of, the trehalase, the function of the phosphate may involve stabilizing the protein conformation and/or initiating protein aggregation. Sodium arsenate was able to substitute to some extent for the sodium phosphate requirement, whereas inorganic pyrophosphate and polyphosphates were inhibitory. The purified trehalase showed a single 71 kDa band on SDS gels, but active enzyme eluted in the void volume of a Sephracryl S-300 column, suggesting a molecular mass of about 1500 kDa or a multimer of 20 or more subunits. The trehalase is highly specific for alpha,alpha-trehalose and did not hydrolyze alpha,beta-trelalose or beta,beta-trehalose, trehalose dimycolate, or any other alpha-glucoside or beta-glucoside. Attempts to obtain a trehalase-negative mutant of M. smegmatis have been unsuccessful, although deletions of other trehalose metabolic enzymes have yielded viable mutants. This suggests that trehalase is an essential enzyme for these organisms. The enzyme has a pH optimum of 7.1, and is active in various buffers, as long as inorganic phosphate and Mg(2+) are present. Glucose was the only product produced by the trehalase in the presence of either phosphate or arsenate.     

Replacement of lysine 269 by arginine in Escherichia coli tryptophan indole-lyase affects the formation and breakdown of quinonoid complexes

Lysine 269 in Escherichia coli tryptophan indole-lyase (tryptophanase) has been changed to arginine by site-directed mutagenesis. The resultant K269R mutant enzyme exhibits kcat values about 10% those of the wild-type enzyme with S-(o-nitrophenyl)-L-cysteine, L-tryptophan, and S-benzyl-L-cysteine, while kcat/Km values are reduced to 2% or less. The pH profile of kcat/Km for S-benzyl-L-cysteine for the mutant enzyme exhibits two pK alpha values which are too close to separate, with an average value of 7.6, while the wild-type enzyme exhibits pK alpha values of 6.0 and 7.8. The pK alpha for the interconversion of the 335 and 412 nm forms of the K269R enzyme is 8.3, while the wild-type enzyme exhibits a pK alpha of 7.4. Steady-state kinetic isotope effects on the reaction of [alpha-2H]S-benzyl-L-cysteine with the K269R mutant enzyme (Dkcat = 2.0; D(kcat/Km) = 3.9) are larger than those of the wild-type enzyme (Dkcat = 1.4; D(kcat/Km) = 2.9). Rapid scanning stopped-flow kinetic studies demonstrate that the K269R mutant enzyme does not accumulate quinonoid intermediates with L-alanine, L-tryptophan, or S-methyl-L-cysteine, but does form quinonoid absorption peaks in complexes with S-benzyl-L-cysteine and oxidolyl-L-alanine, whereas wild-type enzyme forms prominent quinonoid bands with all these amino acids. Single wavelength stopped-flow kinetic studies demonstrate that the alpha-deprotonation of S-benzyl-L-cysteine is 6-fold slower in the K269R mutant enzyme, while the intrinsic deuterium kinetic isotope effect is less for the K269R enzyme (Dk = 4.2) than for the wild-type (Dk = 7.9). The decay of the K269R quinonoid intermediate in the presence of benzimidazole is 7.1-fold slower than that of the wild-type enzyme. These results demonstrate that Lys-269 plays a significant role in the conformational changes or electrostatic effects obligatory to the formation and decomposition of the quinonoid intermediate, although it is not an essential basic residue.     

Activity and Heat Stability of Trehalase from the Mycelium and Ascospores of Neurospora

Trehalases from the ascospores of Neurospora tetrasperma and the mycelium of N. crassa were compared. Enzymes from both sources have identical electrophoretic mobilities, K(m)'s, responses to pH, immunological reactions, and activities in low-molarity buffers. Because both enzymes are so similar, conclusions about the properties of the ascospore enzyme may, be made by studying mycelial trehalase. Mycelial trehalase is most active and stable in low-molarity buffers. The enzyme exists in at least three species; the smallest has a molecular weight between 105,000 and 125,000 and is predominant in low-molarity buffers at 37 C. The stability of trehalase to heating at 65 C can be increased by increasing enzyme concentration and by the addition of polyols. Ascospores contain large amounts of trehalose, which protects trehalase from heat inactivation at 65 C. The importance of this phenomenon in vivo and its relationship to the localization of trehalase in ascospores is discussed.     

The variation of catalytic efficiency of Bacillus cereus metallo-beta-lactamase with different active site metal ions

The kinetics and mechanism of hydrolysis of the native zinc and metal substituted Bacillus cereus (BcII) metallo-beta-lactamase have been investigated. The pH and metal ion dependence of k(cat) and k(cat)/K(m), determined under steady-state conditions, for the cobalt substituted BcII catalyzed hydrolysis of cefoxitin, cephaloridine, and cephalexin indicate that an enzyme residue of apparent pK(a) 6.3 +/- 0.1 is required in its deprotonated form for metal ion binding and catalysis. The k(cat)/K(m) for cefoxitin and cephalexin with cadmium substituted BcII is dependent on two ionizing groups on the enzyme: one of pK(a1) = 8.7 +/- 0.1 required in its deprotonated form and the other of pK(a2) = 9.3 +/- 0.1 required in its protonated form for activity. The pH dependence of the competitive inhibition constant, K(i), for CdBcII with l-captopril indicates that pK(a1) = 8.7 +/- 0.1 corresponds to the cadmium-bound water. For the manganese substituted BcII, the pH dependence of k(cat)/K(m) for benzylpenicillin, cephalexin, and cefoxitin similarly indicated the importance of two catalytic groups: one of pK(a1) = 8.5 +/- 0.1 which needs to be deprotonated and the other of pK(a2) = 9.4 +/- 0.1 which needs to be protonated for catalysis; the pK(a1) was assigned to the manganese-bound water. The rate was metal ion concentration dependent at the highest manganese concentrations used (10(-)(3) M). The metal substituted species have similar or higher catalytic activities compared with the zinc enzyme, albeit at pHs above 7. Interestingly, with cefoxitin, a very poor substrate for ZnBcII, both k(cat) and k(cat)/K(m) increase with increasing pK(a) of the metal-bound water, in the order Zn < Co < Mn < Cd. A higher pK(a) for the metal-bound water for cadmium and manganese BCII leads to more reactive enzymes than the native zinc BcII, suggesting that the role of the metal ion is predominantly to provide the nucleophilic hydroxide, rather than to act as a Lewis acid to polarize the carbonyl group and stabilize the oxyanion tetrahedral intermediate.     

Factorization of the Michaelis functions.

Each Michaelis function that expresses the concentration of one of the species AL2, AL and A in terms of the concentration of free ligand (or its logarithm) is the product of two functions each of which represents the degree of ligation or de-ligation of a single site. These hypothetical sites have pK values of pK (SEE ARTICLE) where pK and alpha are defined by writing the two molecular pK values as pK +/- log2alpha. The factors are thus real if alpha larger than or equal to 1, i.e. if the binding of L by A is not positively co-operative. The dependence of [AL] on 1n[L] is compared with relations that represent other ligand-dependent equilibria.     

The regulatory properties of rabbit muscle pyruvate kinase. The effect of pH.

The regulatory behavior of rabbit pyruvate kinase has been studied as a function of pH. The initial velocity of the enzyme-catalysed reaction as a function of ADP concentration was analysed with the exponential model for a regulatory enzyme. The analysis of the exponential model parameters as functions of pH provided pK values of 6.6 and 8.08 for the free enzyme in its fully ADP-bound conformation. By contrast, the binding of ADP to the ADP-free conformation of the free enzyme did not involve groups that ionize within the pH range (6.2-8.5) of these experiments. The results suggest that homotropic allosteric interactions actually alter the mode of ADP binding. The pK values of 6.63 and 9.00 determined from the analysis of V as a function of pH are readily interpreted in terms of a direct phosphoryl-transfer mechanism in which the beta-phosphoryl group of ADP (pK 6.63) acts as the nucleophile and a lysine epsilon-amino group (pK 9.0) acts as the proton donor in the pyruvate kinase reaction.     

Control of L-ornithine specificity in Escherichia coli ornithine transcarbamoylase. Site-directed mutagenic and pH studies

Escherichia coli ornithine transcarbamoylase displays a strict specificity toward its second substrate L-ornithine. After forming a binary complex with carbamoyl phosphate and undergoing an induced-fit isomerization (Miller, A. W., and Kuo, L. C. (1990) J. Biol. Chem. 265, 15023-15027), the enzyme selects only the minor, zwitterionic ornithine with an uncharged delta-amino group for transcarbamoylation. Formation of the productive ternary complex is linked to two enzymic ionizations (pK alpha 6.2 approximately 6.3 and 9.1 approximately 9.3) and two ornithine ionizations (pK alpha 8.5 and 10.6) (Kuo, L. C., Herzberg, W., and Lipscomb, W. N. (1985) Biochemistry 24, 4754-4761). To elucidate the mechanism through which substrate specificity is achieved, the binding of L-ornithine to two site-specific point mutants (Arg-57----Gly and Cys-273----Ala) of the enzyme has been examined. For the Gly-57 mutant enzyme, which does not undergo the induced-fit isomerization, affinity for ornithine drops by a factor of 500. The pH profile of the apparent equilibrium constant governing the association of L-ornithine to the binary complex of this mutant reveals that only two enzymic ionizations affect ornithine binding. The ionizations linked to L-ornithine are not detected. Hence, the preisomerized binary complex binds not only poorly but also indiscriminately all ionic species of L-ornithine. For the Ala-273 mutant enzyme, which exhibits the induced-fit isomerization, affinity of the amino acid is decreased by an order of magnitude. Ionizations of L-ornithine to yield a zwitterion for binding are detected in pH analyses for this mutant, but the pK alpha of 6.2 associated with the enzymic deprotonation in the wild type is absent. Therefore, Cys-273 is a binding site of L-ornithine. The D-isomer of ornithine is a very weak, deadend ligand to all three forms of the enzyme with affinities in the millimolar range. Employing the estimated affinities of D- and L-ornithine, the binding stereospecificity of the wild-type and mutant binary complexes toward the amino acid substrate may be evaluated. L-Ornithine binds preferentially over D-ornithine by two and four orders of magnitude in the absence and presence of protein isomerization, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)     

1H nuclear-magnetic-resonance studies of porcine lutropin and its alpha and beta subunits.

The titration curves of the histidine residues of porcine lutropin and its isolated alpha and beta subunits have been determined by following the pH-dependence of the imidazole C-2 proton resonances. The isolated alpha subunit contains a buried histidine, whose C-2 proton does not exchange with solvent, and which has the unusually low pK of 3.3. In the native hormone all the histidine residues have relatively normal pK values (between 5.7 and 6.2). The four histidine C-2 proton resonances have been assigned to specific residues in the amino-acid sequence, by means of deuterium and tritium exchange experiments on the alpha subunit and its des(92-96) derivative. The histidine with a pK of 3.3 is identified as His-alpha87. The effects of pH on tyrosine and methyl proton resonances show that the titration of His-87 in the isolated alpha subunit is accompanied by a significant conformational change which involves loosening of the protein structure but which is not a normal unfolding transition. The role of conformational changes in the generation of biological activity by subunit association in the glycoprotein hormones is discussed.     

Solubilization and characterization of a cell wall-bound trehalase from ascospores of the fission yeast Schizosaccharomyces pombe

The genome of the fission yeast Schizosaccharomyces pombe lacks sequence homologs to ath1 genes coding for acid trehalases in other yeasts or filamentous fungi. However, acid trehalase activity is present at the spore stage in the life cycle of the fission yeast. The enzyme responsible for this activity behaves as a surface enzyme covalently linked to the spore cell walls in both wild-type and ntp1 mutant strains devoid of neutral trehalase. Lytic treatment of particulated cell wall fractions allowed the solubilization of the enzyme into an active form. We have characterized this soluble enzyme and found that its kinetic parameters, optimum pH and temperature, thermal denaturation and salt responses are closely similar to other conventional acid trehalases. Hence, this rather unusual enzyme can be recognized as acid trehalase by its biochemical properties although it does not share genetic homology with other known acid trehalases. The potential role of such acid trehalase in the mobilization of trehalose is discussed.     

Partial purification and characterization of trehalase from axenically grown myxamoebae of Dictyostelium discoideum

Lysosomal trehalase from the myxamoebae of Dictyostelium discoideum has been partially purified. The behavior of the enzyme under different chromatographic and electrophoretic conditions reveals its close similarities to other lysosomal enzymes that have been studied earlier. The cellular trehalase, which is electrophoretically homogeneous, appears as two peaks of activity when subjected to hydroxyapatite and gel filtration chromatography. The enzyme has isoelectric points of 4.0 and less than 2.5. Among natural disaccharides tested, the purified trehalase showed absolute specificity for trehalose with an apparent Km of 1.15 mM. However, the enzyme efficiently utilized the synthetic sugar alpha-D-glucosyl fluoride as a substrate. Various methods were employed to estimate the apparent molecular weight, which was found to lie in the range of 30-162 kDa.     

Protein phosphorylation and trehalase activation in Candida utilis

Abstract Candida utilis cells contain a regulatory trehalase enzyme (280 kDa) which can be activated by cAMP-dependent phosphorylation. A 100-fold purification of this enzyme activity results in the enrichment of a protein band of apparent M _r 70 000 as identified by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). This component is phosphorylated in vivo under conditions in which trehalase activation occurs in whole cells. It is concluded that the trehalase enzyme might be a tetramer, composed of 4 identical 70-kDa subunits.     

Evidence that cysteine 298 is in the active site of tryptophan indole-lyase

Escherichia coli tryptophan indole-lyase (tryptophanase) mutants, with cysteine residues 294 and 298 selectively replaced by serines, have been prepared by site-directed mutagenesis. Both mutant enzymes are highly active for beta-elimination reactions measured with both L-tryptophan and S-(o-nitrophenyl)-L-cysteine. The Cys-294----Ser mutant enzyme is virtually identical to the wild type with respect to pyridoxal phosphate binding (KCO = 2 microM), cofactor absorption spectrum (lambda max = 420 and 337 nm) and pH dependence (pK alpha = 7.3), pH profile for catalysis, and rate of bromopyruvic acid inactivation. In contrast, the Cys-298----Ser mutant enzyme exhibits a reduced affinity for pyridoxal phosphate (KCO = 6 microM), a shift in the cofactor absorption spectrum to 414 nm and an altered pK alpha = 8.5, an alkaline shift in the pH profile for catalysis, and resistance to inactivation of the apoenzyme by bromopyruvic acid. The C298S mutant enzyme (wherein cysteine 298 is altered to serine) also undergoes an isomerization to an unreactive state upon storage at 4 degrees C. These results demonstrate that the sulfhydryl groups of Cys-294 and Cys-298 are catalytically nonessential. However, these data suggest that Cys-298 is located within or very near the active site of the enzyme and is the reactive cysteine residue previously observed by others.     

Solvent isotope effects and the pH dependence of laccase activity under steady-state conditions

Solvent isotope effects and the pH dependence of laccase catalysis under steady-state conditions were examined with a rapid reductant to assess the potential roles of protein protic groups and the catalytic mechanism. The pH dependence of both reductant-dependent and reductant-independent steps showed bell-shaped profiles implicating at least two protic groups in each case. The apparent pKa values were: for the reductant-independent step(s), pK alpha 1 = 8.98 +/- 0.02 and pK alpha 2 = 5.91 +/- 0.03; for the reductant-dependent step(s), pK' alpha 1 = 7.55 +/- 0.12, pK' alpha 2 = 8.40 +/- 0.23. No solvent isotope effect on reductant-dependent steps was detected other than a standard shift effect. However, a significant solvent isotope effect on a reductant-independent step(s) was observed; kH/kD = 2.12 at the pH optimum of 7.5. The concentration dependence of the D2O effect indicated that a single proton was involved. Simulations of the p(H,D) data suggested that the solvent isotope effect was associated with the protein protic group required in its undissociated form (pK alpha 2). The pH effects on reductant-dependent steps are apparently associated with reductant-dependent steps that occur between O2 binding and water formation in the catalytic reaction sequence.     

The role of carboxyl, guanidine and imidazole groups in catalysis by a midgut trehalase purified from an insect larvae

A trehalase (EC 3.2.1.28) of 67 kDa was purified to homogeneity from the midgut of Spodoptera frugiperda (Lepidoptera) larvae. The enzyme is inhibited by toxic beta-glucosides produced by plants (amygdalin, prunasin, salicin and phlorezin) and by their aglycones (mandelonitrile, phloretin). From kcat and Km values determined in different pHs, the pKa values of catalytic essential groups were calculated (pKa = 4.5 and pKa = 8.0). These pKa values agree with the ones determined from enzyme chemical in activation with carbodiimide and phenyl glyoxal, respectively, indicating that the enzyme has a carboxyl group that act as a nucleophile and a guanidine group that is the proton donor during the catalytic cycle. The enzyme has two putative subsites for glucose binding. Based on the protection afforded by ligands against chemical modification, the roles of the subsites were inferred. Thus, the one that binds the competitive inhibitors, methyl alpha-glucoside (MalphaGlu) and mandelonitrile, contains the catalytic carboxyl, whereas the other having the catalytic Arg residue binds the competitive inhibitor Tris. Diethyl pyrocarbonate is ineffective except in the presence of MalphaGlu, when it decreases trehalase activity and changes the pKa value of the catalytic Arg residue. This suggests that the pKa value of the Arg residue is modulated by a His residue located near the active site. This also indicates that the enzyme molecule changes its conformation when the subsite containing the carboxyl group is occupied. The increase in trehalase inactivation by phenyl glyoxal in the presence of MalphaGlu agrees with the last observation.