Identification of histidine residues at the active site of Megalobatrachus japonicus alkaline phosphatase by chemical modification

Alkaline phosphatase from Megalobatrachus japonicus was inactivated by diethyl pyrocarbonate (DEP). The inactivation followed pseudo-first-order kinetics with a second-order rate constant of 176 M(-1) x min(-1) at pH 6.2 and 25 degrees C. The loss of enzyme activity was accompanied with an increase in absorbance at 242 nm and the inactivated enzyme was re-activated by hydroxylamine, indicating the modification of histidine residues. This conclusion was also confirmed by the pH profiles of inactivation, which showed the involvement of a residue with pK(a) of 6.6. The presence of glycerol 3-phosphate, AMP and phosphate protected the enzyme against inactivation. The results revealed that the histidine residues modified by DEP were located at the active site. Spectrophotometric quantification of modified residues showed that modification of two histidine residues per active site led to complete inactivation, but kinetic stoichiometry indicated that one molecule of modifier reacted with one active site during inactivation, probably suggesting that two essential histidine residues per active site are necessary for complete activity whereas modification of a single histidine residue per active site is enough to result in inactivation.     

Evidence for the presence of a critical histidine residue at the active site in glyceraldehyde-3-phosphate dehydrogenase of Ehrlich ascites carcinoma cells

Ehrlich ascites carcinoma (EAC) cell glyceraldehyde-3-phosphate dehydrogenase (GA3PD) (EC. 1.2.1.12) was completely inactivated by diethyl pyrocarbonate (DEPC), a fairly specific reagent for histidine residues in the pH range of 6.0-7.5. The rate of inactivation was dependent on pH and followed pseudo-first order reaction kinetics. The difference spectrum of the inactivated and native enzymes showed an increase in the absorption maximum at 242 nm, indicating the modification of histidine residues. Statistical analysis of the residual enzyme activity and the extent of modification indicated modification of one essential histidine residue to be responsible for loss of the catalytic activity of EAC cell GA3PD. DEPC inactivation was protected by substrates, D-glyceraldehyde-3-phosphate and NAD, indicating the presence of essential histidine residue at the substrate-binding region of the active site. Double inhibition studies also provide evidence for the presence of histidine residue at the active site.     

The effect of histidine modification on the activity of myo-inositol monophosphatase from bovine brain.

The pH dependence of myo-inositol monophosphatase may indicate a role for histidine residues in the catalytic mechanism (Ganzhorn, A. J., and Chanal, M.-C. (1990) Biochemistry 29, 6065-6071). This possibility was investigated by chemical modification. At pH 6.0 and 25 degrees C, the enzyme was inactivated by diethylpyrocarbonate in a pseudo-first order reaction with a bimolecular rate constant of 0.37 M-1 s-1. Two histidines were modified rapidly with no effect on enzyme activity, while 3 residues were modified at a slower rate corresponding to the rate of inactivation. No noticeable changes in the secondary structure of the enzyme were observed by comparison of circular dichroic spectra before and after modification. Treatment of myo-inositol monophosphatase with diethylpyrocarbonate in the presence of inositol 1-phosphate, Mg2+, and Li+ protected 2 residues from modification and decreased the inactivation rate by about 5-fold. Spectrophotometric analysis, the restoration of enzyme activity by hydroxylamine, and the lack of any inhibitory effect with alkylating agents suggest that inactivation is due solely to modification of histidine. We conclude that a histidine residue is essential for activity and may act as a base catalyst during hydrolysis of the substrate.     

Evidence for an essential histidine in neutral endopeptidase 24.11

Rat kidney neutral endopeptidase 24.11, "enkephalinase", was rapidly inactivated by diethyl pyrocarbonate under mildly acidic conditions. The pH dependence of inactivation revealed the modification of an essential residue with a pKa of 6.1. The reaction of the unprotonated group with diethyl pyrocarbonate exhibited a second-order rate constant of 11.6 M-1 s-1 and was accompanied by an increase in absorbance at 240 nm. Treatment of the inactivated enzyme with 50 mM hydroxylamine completely restored enzyme activity. These findings indicate histidine modification by diethyl pyrocarbonate. Comparison of the rate of inactivation with the increase in absorbance at 240 nm revealed a single histidine residue essential for catalysis. The presence of this histidine at the active site was indicated by (a) the protection of enzyme from inactivation provided by substrate and (b) the protection by the specific inhibitor phosphoramidon of one histidine residue from modification as determined spectrally. The dependence of the kinetic parameter Vmax/Km upon pH revealed two essential residues with pKa values of 5.9 and 7.3. It is proposed that the residue having a kinetic pKa of 5.9 is the histidine modified by diethyl pyrocarbonate and that this residue participates in general acid/base catalysis during substrate hydrolysis by neutral endopeptidase 24.11.     

Modification of uridine phosphorylase from Escherichia coli by diethyl pyrocarbonate. Evidence for a histidine residue in the active site of the enzyme.

Uridine phosphorylase from Escherichia coli is inactivated by diethyl pyrocarbonate at pH 7.1 and 10 degrees C with a second-order rate constant of 840 M-1.min-1. The rate of inactivation increases with pH, suggesting participation of an amino acid residue with pK 6.6. Hydroxylamine added to the inactivated enzyme restores the activity. Three histidine residues per enzyme subunit are modified by diethyl pyrocarbonate. Kinetic and statistical analyses of the residual enzymic activity, as well as the number of modified histidine residues, indicate that, among the three modifiable residues, only one is essential for enzyme activity. The reactivity of this histidine residue exceeded 10-fold the reactivity of the other two residues. Uridine, though at high concentration, protects the enzyme against inactivation and the very reactive histidine residue against modification. Thus it may be concluded that uridine phosphorylase contains only one histidine residue in each of its six subunits that is essential for enzyme activity.     

Inactivation of Saccharomyces cerevisiae glucose-6-phosphate dehydrogenase by diethylpyrocarbonate

Glucose-6-phosphate dehydrogenase purified from Saccharomyces cerevisiae is rapidly inactivated by diethylpyrocarbonate at pH 6.8 and 30 degrees C with a concomitant increase in absorbance at 242 nm. The second-order rate constant for inactivation was calculated to be 487.8 M-1 min-1. The pH dependence of inactivation suggests the involvement of an amino acid residue having a pKa of 6.77. These results indicate that the inactivation is due to the modification of a histidine residue(s). In the presence of substrate, glucose-6-phosphate or NADP+, the rate of inactivation is decreased, indicating that the essential histidine residue(s) is located at the active site, possibly at the region of overlap of substrates at the binding site.     

Ethoxyformylation of ribonuclease U2 form Ustilago sphaerogena.

RNase U2 was inactivated by incubation with ethoxyformic anhydride at pH 6.0 and pH 4.5. The absorbance of the RNase U2 increased at around 250 nm and decreased at around 280 nm. The inactivation occurred in parallel with the amount of modified histidine and plots of the relationship between the remaining activity and the modified histidine suggested that the modification of one of the two histidine residues totally inactivated the enzyme. The inactivated enzyme RNase U2 was reactivated by a low concentration of hydroxyamine, with removal of the ethoxyformyl group from the modified histidine residue. At pH 4.5, 2'-adenylate and 2'-guanylate protected RNase U2 from inactivation by ethoxyformic anhydride. The difference CD spectra showed that the ability of RNase U2 to form a complex with 2'-adenylate was lost on ethoxyformylation.     

Essential histidine residues in lysolecithin:lysolecithin acetyltransferase from rabbit lung

Both activities of rabbit lung lysolecithin:lysolecithin acyltransferase (EC 3.1.1.5), hydrolysis and transacylation, are inactivated by diethylpyrocarbonate. The reaction follows pseudo-first-order kinetics, and second-order rate constants of 1.17 mM-1min-1 for hydrolysis and 0.56 mM-1 min-1 for transacylation were obtained at pH 6.5 and 37 degrees C. The rate of inactivation is dependent on pH, showing the involvement of a group with a pK of 6.5. The difference spectra showed an increase in absorbance at 242 nm, indicating the modification of histidine residues. The activity lost by diethylpyrocarbonate modification can be partially recovered by hydroxylamine treatment. The statistical analysis of residual fractional activity versus the number of modified histidine residues leads to the conclusion that two histidine residues are essential for the hydrolytic activity, whereas transacylation activity depends on only one essential histidine. The substrate and substrate analogs protected the enzyme against inactivation by diethylpyrocarbonate, suggesting that the essential residues are located at or near the active site of the enzyme.     

Chemical modification by diethylpyrocarbonate of an essential histidine residue in 3-ketovalidoxylamine A C-N lyase

3-Ketovalidoxylamine A C-N lyase of Flavobacterium saccharophilum is a monomeric protein with a molecular weight of 36,000. Amino acid analysis revealed that the enzyme contains 5 histidine residues and no cysteine residue. The enzyme was inactivated by diethylpyrocarbonate (DEP) following pseudo-first order kinetics. Upon treatment of the inactivated enzyme with hydroxylamine, the enzyme activity was completely restored. The difference absorption spectrum of the modified versus native enzyme exhibited a prominent peak around 240 nm, but there was no absorbance change above 270 nm. The pH-dependence of inactivation suggested the involvement of an amino acid residue having a pKa of 6.8. These results indicate that the inactivation is due to the modification of histidine residues. Substrates of the lyase, p-nitrophenyl-3-ketovalidamine, p-nitrophenyl-alpha-D-3-ketoglucoside, and methyl-alpha-D-3-ketoglucoside, protected the enzyme against the inactivation, suggesting that the modification occurred at or near the active site. Although several histidine residues were modified by DEP, a plot of log (reciprocal of the half-time of inactivation) versus log (concentration of DEP) suggested that one histidine residue has an essential role in catalysis.     

Chemical modification of S-adenosylhomocysteinase by a water-soluble carbodiimide

S-Adenosylhomocysteinase (EC 3.3.1.1) from rat liver is inactivated by 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate (CMC) in a pseudo-first-order fashion. The rate of inactivation is linearly related to the concentration of the reagent, and a second-order rate constant of 4.94 +/- 0.27 M-1 min-1 is obtained at pH 5.5 and 25 degrees C. The inactivation does not involve change in the quaternary structure of the enzyme nor modification or release of the enzyme-bound NAD. Lack of modification at tyrosine, serine, cysteine, histidine, and lysine residues and the fact that the inactivation is favored at low pH suggest that the inactivation is caused by the modification of a carboxyl group. Statistical analysis of the relationship between the residual enzyme activity and the extent of modification, and comparison of the number of residues modified in the presence and absence of the substrate adenosine show that, among four reactive residues per enzyme subunit, only one residue which reacts more rapidly with the reagent than the rest is critical for activity. The CMC-modified enzyme binds adenosine and S-adenosylhomocysteine and is able to oxidize the 3' hydroxyl of these substrates, but apparently fails to catalyze the abstraction of the 4' proton of adenosine.     

Chemical modification of essential histidine residues in aspartase with diethylpyrocarbonate

Aspartase purified from Escherichia coli W cells was inactivated by diethylpyrocarbonate following pseudo-first order kinetics. Upon treatment of the inactivated enzyme with NH2OH, the enzyme activity was completely restored. The difference absorption spectrum of the modified vs. native enzyme preparations exhibited a prominent peak around 240 nm. The pH-dependence of the inactivation rate suggested that an amino acid residue having a pK value of 6.6 was involved in the inactivation. These results indicate that the inactivation was due to the modification of histidine residues. L-Aspartate and fumarate, substrates for the enzyme, and the Cl- ion, an inhibitor, protected the enzyme against the inactivation. Inspection of the spectral change at 240 nm associated with the inactivation in the presence and absence of the Cl- ion revealed that the number of histidine residues essential for the enzyme activity was less than two. Partial inactivation did not result in an appreciable change in the substrate saturation profiles. These results suggest that one or two histidine residues are located at the active site of aspartase and participate in an essential step in the catalytic reaction.     

Chemical modification of NADP-isocitrate dehydrogenase from Cephalosporium acremonium evidence of essential histidine and lysine groups at the active site.

NADP-isocitrate dehydrogenase from Cephalosporium acremonium CW-19 has been inactivated by diethyl pyrocarbonate following a first-order process giving a second-order rate constant of 3.0 m-1. s-1 at pH 6.5 and 25 degrees C. The pH-inactivation rate data indicated the participation of a group with a pK value of 6.9. Quantifying the increase in absorbance at 240 nm showed that six histidine residues per subunit were modified during total inactivation, only one of which was essential for catalysis, and substrate protection analysis would seem to indicate its location at the substrate binding site. The enzyme was not inactivated by 5, 5'-dithiobis(2-nitrobenzoate), N-ethylmaleimide or iodoacetate, which would point to the absence of an essential reactive cysteine residue at the active site. Pyridoxal 5'-phosphate reversibly inactivated the enzyme at pH 7.7 and 5 degrees C, with enzyme activity declining to an equilibrium value within 15 min. The remaining activity depended on the modifier concentration up to about 2 mm. The kinetic analysis of inactivation and reactivation rate data is consistent with a reversible two-step inactivation mechanism with formation of a noncovalent enzyme-pyridoxal 5'-phosphate complex prior to Schiff base formation with a probable lysyl residue of the enzyme. The analysis of substrate protection shows the essential residue(s) to be at the active site of the enzyme and probably to be involved in catalysis.     

An essential histidine residue in the catalytic mechanism of mammalian glutathione reductase

Glutathione reductase from human erythrocytes was inactivated by ethoxyformic anhydride, and > 95% activity was lost by modification of about 1–1.5 histidine residues per flavin (or subunit), as measured by the increased absorbance at 240 nm. Full reactivation was obtained with hydroxylamine. The rate of inactivation increased with pH and an apparent pK = 5.9 was obtained for the protolytic dissociation. The modified enzyme was inactive with NADPH and GSSG as substrates, but almost fully active in catalysis of a transhydrogenase reaction involving pyridine nucleotides. The visible absorption spectrum of oxidized or two-electron-reduced enzyme was not changed, but the flavin fluorescence of oxidized enzyme increased 2-fold after the modification. NADPH or NADP⁺ did not protect the enzyme against inactivation. It is concluded that the modification affects a histidine involved in the second half-reaction of the catalysis, i.e. reduction of GSSG by the dithiol of reduced enzyme. Glutathione reductase from three additional mammalian sources was similarly inactivated, but enzyme from yeast was much less inactivated by the corresponding treatment with ethoxyformic anhydride.     

Essential histidine residue in 3-ketosteroid-delta 1-dehydrogenase.

The variation with pH of kinetic parameters was examined for 3-ketosteroid-delta 1-dehydrogenase from Nocardia corallina. The Vmax/Km profile for 4-androstenedione indicates that activity is lost upon protonation of a cationic acid-type group with a pK value of 7.7. The enzyme was inactivated by diethylpyrocarbonate at pH 7.4 and the inactivation was substantially prevented by androstadienedione. Analyses of reactivation with neutral hydroxylamine, pH variation, and spectral changes of the inactivated enzyme revealed that the inactivation arises from modification of a histidine residue. Studies with [14C]diethylpyrocarbonate provided support for the idea that the 1-2 essential histidine residues are essential for the catalytic activity of the enzyme. Dye-sensitized photooxidation led to 50% inactivation of the enzyme with the decomposition of two histidine residues. This inactivation was also prevented by androstadienedione. Dancyl chloride caused a loss of the enzyme activity. Modifiers of glutamic acid, aspartic acid, cysteine, and lysine did not affect the enzyme activity. Butanedione and phenylglyoxal in the presence of borate rapidly inactivated the enzyme, indicating that arginine residues also have a crucial function in the active site. The data described support the previously proposed mechanism of beta-oxidation of 3-ketosteroid.     

Histidine 268 in 3-deoxy-D-arabino-heptulosonic acid 7-phosphate synthase plays the same role as histidine 202 in 3-deoxy-D-manno-octulosonic acid 8-phosphate synthase

The enzyme 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAH 7-P) synthase (Phe) is inactivated by diethyl pyrocarbonate (DEPC). The inactivation is first order with respect to enzyme and DEPC concentrations with a pseudo-second order rate constant of inactivation by DEPC of 4.9 +/- 0.8 m(-1) s(-1) at pH 6.8 and 4 degrees C. The dependence of inactivation on pH and the spectral features of enzyme modified at specific pH values imply that both histidine and cysteine residues are modified, which is confirmed by site-directed mutagenesis. Analysis of the chemical modification data indicates that one histidine is essential for activity. DAH 7-P synthase (Phe) is protected against DEPC inactivation by phosphoenolpyruvate, whereas d-erythrose 4-phosphate offers only minimal protection. The conserved residues H-172, H-207, H-268, and H-304 were individually mutated to glycine. The H304G and H207G mutants retain some level of activity, whereas the H268G and H172G mutants are virtually inactive. A comparison of the circular dichroism spectra of wild-type enzyme and the various mutants demonstrates that H-172 may play a structural role. Comparison of the UV spectra of the H268G and wild-type enzymes saturated with Cu(2+) indicates that the metal-binding site of the H268G mutant resembles that of the wild-type enzyme. The residue H-268 may play a catalytic role based on the site-directed mutagenesis and spectroscopic studies. Cysteine 61 appears to influence the pK(a) of H-268 in the wild-type enzyme. The pK(a) of H-268 increases from 6.0 to 7.0 following mutation of C-61 to glycine.     

Aspartokinase-homoserine dehydrogenase I from Escherichia coli: pH and chemical modification studies of the kinase activity

The pH variation of the kinetic parameters was examined for the kinase activity of the bifunctional enzyme aspartokinase--homoserine dehydrogenase I isolated from Escherichia coli. The V/K profile for L-aspartic acid indicates the loss of activity upon protonation of a cationic acid type group with a pK value near neutrality. Incubation of the enzyme with diethyl pyrocarbonate at pH 6.0 results in a loss of enzymic activity. The reversal of this reaction by neutral hydroxylamine, the appearance of a peak at 242 nm for the inactivated enzyme, and the observation of a pK value of 7.0 obtained from variation of the inactivation rate with pH all suggest that enzyme inactivation occurs by modification of histidine residues. The substrate L-aspartic acid protects one residue against inactivation, which implies that this histidine may participate in substrate binding or catalysis. Activity loss was also observed at high pH due to the ionization of a neutral acid group with a pK value of 9.8. The reactions of AK-HSD I with N-acetylimidazole and tetranitromethane have been investigated to obtain information about the functional role of tyrosyl residues in the enzyme. The acylation of tyrosines leads to inactivation of the enzyme, which can then be fully reversed by treatment with hydroxylamine. Incubation of the enzyme with tetranitromethane at pH 9.5 also leads to rapid inactivation, and the substrates of the kinase reaction provide substantial protection against inactivation. However, three tyrosines are protected by substrates, implying a structural role for these amino acids.     

Inactivation of Acinetobacter calcoaceticus acetate kinase by diethylpyrocarbonate

Acetate kinase purified from Acinetobacter calcoaceticus was inhibited by diethylpyrocarbonate with a second-order rate constant of 620 M-1.min-1 at pH 7.4 at 30 degrees C and showed a concomitant increase in absorbance at 240 nm due to the formation of N-carbethoxyhistidyl derivative. Activity could be restored by hydroxylamine and the pH curve of inactivation indicates the involvement of a residue with a pKa of 6.64. Complete inactivation of acetate kinase required the modification of seven residues per molecule of enzyme. Statistical analysis showed that among the seven modifiable residues, only one is essential for activity. 5,5'-dithiobis(2-nitrobenzoic acid), p-chloromercuryphenylsulfonate, N-ethylmaleimide and phenylglyoxal did not affect the enzyme activity. These results suggest that the inactivation is due to the modification of one histidine residue. The substrates, acetate and ATP, protected the enzyme against inactivation, indicating that the modified histidine residue is located at or near the active site.     

Reaction of Ascaris suum phosphofructokinase with diethylpyrocarbonate. Inactivation and desensitization to allosteric modulation

Reaction of the phosphofructokinase from Ascaris suum with the reagent, diethylpyrocarbonate (DEPC), results in the loss of enzymatic activity. Treatment of the inactivated enzyme with hydroxylamine brings about the recovery of almost 80% of the original activity suggesting that the modified residues are histidines. Further evidence for the modification of histidines is that concomitant with the loss of activity, there is a change in A242 nm that corresponds to the derivatization of 5-6 histidines per subunit. There is no change in A278 nm during the derivatization process, thereby ruling out the modification of tyrosines by DEPC. Analyses of the first order inactivation rate constant for DEPC derivatization at different pH values resulted in the determination of a pKa of 6.4 +/- 0.1 for the group on the enzyme that reacts with DEPC. Derivatization of the enzyme with DEPC in the presence of fructose 6-phosphate (Fru-6-P) protected the enzyme against inactivation by 80%. ATP or MgATP gave no protection against DEPC inactivation. When the Fru-6-P-protected enzyme was further reacted with DEPC in the absence of Fru-6-P, a total of 2 histidines were modified per subunit, and the derivatization of one of these could be correlated with activity loss. When the phosphofructokinase that had been derivatized by DEPC in the presence of Fru-6-P was assayed, it was found that it no longer exhibited allosteric properties and appeared to be desensitized to ATP inhibition. This loss of ATP inhibition could be correlated with the modification of 2 histidines per subunit by DEPC. The first order rate constant for desensitization was determined at different pH values and a pKa value of 7.0 +/- 0.2 was obtained for the group(s) responsible for the desensitization. Regulatory studies with the desensitized enzyme revealed that the enzyme was not stimulated by AMP, NH4+, K+, phosphate, sulfate, or hexose bisphosphates. It is concluded that histidine may be involved both in the active site and the ATP inhibitory site of the ascarid phosphofructokinase.     

Chemical modification of sheep-liver 6-phosphogluconate dehydrogenase by diethylpyrocarbonate. Evidence for an essential histidine residue

Sheep liver 6-phosphogluconate dehydrogenase is shown to be inactivated by diethylpyrocarbonate in a biphasic manner at pH 6.0, 25 degrees C. After allowing for the hydrolysis of the reagent, rate constants of 56 M-1 s-1 and 11.0 M-1 s-1 were estimated for the two processes. The complete reactivation of partially inactivated enzyme by neutral hydroxylamine, the elimination of the possibility that modification of cysteine or tyrosine residues are responsible for inactivation, and the magnitudes of the rate constants for inactivation relative to the experimentally determined value for the reaction of diethylpyrocarbonate with N alpha-acetylhistidine (2.2 M-1 s-1), all suggested that enzyme inactivation occurs solely by modification of histidine residues. Comparison of the experimental plot of residual fractional activity versus the number of modified histidine residues per subunit with simulated plots for three hypothetical models, each predicting biphasic kinetics, indicated that inactivation results from the modification of at most one essential histidine residue per subunit, although it appears that other (non-essential) histidines react independently. This histidine is thought to be His-242 and is present in the active site. Evidence in support of its role in catalysis is briefly discussed. Both 6-phosphogluconate and organic phosphate protect against inactivation, and a kinetic analysis of the protection indicated a dissociation constant of 2.1 X 10(-6) M for the enzyme--6-phosphogluconate complex. NADP+ also protected, but this might be due, at least in part, to a reduction in the effective concentration of diethylpyrocarbonate.     

Essential histidine residues in dextransucrase: chemical modification by diethyl pyrocarbonate and dye photo-oxidation

Treatment of Leuconostoc mesenteroides B-512F dextransucrase with diethyl pyrocarbonate (DEP) at pH 6.0 and 25 degrees or photo-oxidation in the presence of Rose Bengal or Methylene Blue at pH 6.0 and 25 degrees, caused a rapid decrease of enzyme activity. Both types of inactivation followed pseudo-first-order kinetics. Enzyme partially inactivated by DEP could be completely reactivated by treatment with 100 mM hydroxylamine at pH 7 and 4 degrees. The presence of dextran partially protected the enzyme from inactivation. At pH 7 or below, DEP is relatively specific for the modification of histidine. DEP-modified enzyme showed an increased absorbance at 240 nm, indicating the presence of (ethoxyformyl)ated histidine residues. DEP modification of the sulfhydryl group of cysteine and of the phenolic group of tyrosine was ruled out by showing that native and DEP-modified enzyme had the same number of sulfhydryl and phenolic groups. DEP modification of the epsilon-amino group of lysine was ruled out by reaction at pH 6 and reactivation with hydroxylamine, which has no effect on DEP-modified epsilon-amino groups. The photo-oxidized enzyme showed a characteristic increase in absorbance at 250 nm, also indicating that histidine had been oxidized, and no decrease in the absorbance at 280 nm, indicating that tyrosine and tryptophan were not oxidized. A statistical, kinetic analysis of the data on inactivation by DEP showed that two histidine residues are essential for the enzyme activity. Previously, it was proposed that two nucleophiles at the active site attack bound sucrose, to give two covalent D-glucosyl-enzyme intermediates. We now propose that in addition, two imidazolium groups of histidine at the active site donate protons to the leaving, D-fructosyl moieties. The resulting imidazole groups then facilitate the formation of the alpha-(1----6)-glycosidic linkage by abstracting protons from the C-6-OH groups, and become reprotonated for the next series of reactions.