Amino acid sequences around 1, 2-epoxy-3-(p-nitrophenoxy)propane-reactive residues in rhizopus chinensis acid protease: homology with pepsin and rennin.

Two different peptides containing an aspartyl residue reactive with 1, 2-epoxy-3-(p-nitrophenoxy)propane (EPNP) in the acid protease from Rhizopus chinensis were isolated from a peptic digest of the EPNP-modified enzyme. One of the peptides was sequenced as Asp-Thr-Gly-Ser-Asp. The amino acid sequence had very high homology with those around the EPNP-reactive aspartyl residues in rennin (chymosin) [EC 3.4.23.4] and pepsin [EC 3.4.23.1]. The other peptide contained no methionine residue and gave the sequence: Asp-Thr-Gly-Thr-Thr-Leu. The N-terminal aspartyl residue of each peptide was deduced to be the EPNP-reactive site.     

Functionally important residues of glutamic acid in E. coli pyrophosphatase. I. Chemical modification and localization in the primary structure]

Inorganic pyrophosphatase of E. coli is rapidly and irreversibly inactivated by 5-ethyl-5-phenylisoxazolium-3'-sulfonate (Woodward's reagent K). The appearance in the absorption spectrum of a maximum at 340 nm testifies to the formation of an enzyme enol ester with the inhibitor. The non-hydrolyzable substrate analog CaPP1 partly protects the enzyme from inactivation. A peptide has been isolated from a tryptic hydrolysate of inactivated enzyme which contains an amino acid residue whose modification is critical for the enzyme activity. This peptide corresponds to residues 95-104 of pyrophosphatase and contains four dicarboxylic acid residues. A peptide containing a modified glutamic acid residue was isolated from modified pyrophosphatase hydrolyzed by protease v8. This peptide represents a fragment of a tryptic modified peptide and has a Glu-Ala-Gly-Glu (residues 98-1C1) structure. It is concluded that inactivation of E. coli pyrophosphatase by Woodward's reagent K is a result of selective modification of Glu98, apparently by the most reactive dicarboxylic amino acid within the enzyme active center.     

The structure and function of acid proteases. IV. Inactivation of the acid protease from Mucor pusillus by acid protease-specific inhibitors.

Mucor pusillus acid protease was rapidly inactivated with 1 : 1 stoichiometry by reaction with diazoacetyl-DL-norleucine methyl ester (DAN) in the presence of cupric ions. Cupric ions were essential for this inactivation. The rate of inactivation was maximal at around pH 6 when the enzyme was mixed with DAN and cupric ions without prior mixing of the reagents, and at pH 5.3 when DAN and cupric ions were mixed and incubated before addition to the enzyme solution. In both cases, the rate of inactivation decreased as the pH was either increased or decreased. The amino acid composition of an acid hydrolysate of the DAN-Modified enzyme was indistinguishable from that of the native enzyme except for the incorporation of about one norleucine residue per molecule of protein. The enzyme was also inactivated by reaction with 1,2-epoxy-3-(p-nitrophenoxy)-propane (EPNP). At the stage of about 90% inactivation, 1.50 residues of EPNP were incorporated per molecule of protein and the rate of inactivation followed pseudo-first order kinetics. The optimal pH for the inactivation was pH 3.0 and the rate of inactivation decreased as the pH was either increased or decreased. Furthermore, the enzyme was strongly inhibited by pepstatin, and the reactions of DAN and of EPNP was also inhibited significantly by prior treatment of the enzyme with pepstatin. These results suggest that the enzyme may have two essential carboxyl groups at the active site, one reactive with DAN in the presence of cupric ions and the other with EPNP, and that pepstatin binds part of the active site to inhibit the reactions with DAN and EPNP as well as the enzyme activity.     

The structure and function of acid proteases. VI. Effects of acid protease-specific inhibitors on the acid proteases from Aspergillus niger var. macrosporus.

1. The Type B acid protease from Aspergillus niger var. macrosporus was inactivated by reaction with diazoacetyl-DL-norleucine methyl ester (DAN), DL-1-diazo-3-tosylamido-2-heptanone (DTH), and L-1-diazo-3-tosylamido-4-phenyl-2-butanone (DTPB) in the presence of cupric ions. The reaction with DAN took place with 1:1 stoichiometry. The enzyme was also inactivated by reaction with 1, 2-epoxy-3-(p-nitrophenoxy)-propane (EPNP) with concomitant incorporation of approximately two EPNP molecules per molecule of protein. Moreover, these reactions of DAN and of EPNP were markedly inhibited by pepstatin. These results seem to indicate that, as in the case of porcine pepsin [EC 3.4.23.1] and related acid proteases, the enzyme has two essential carboxyl groups at the active site, one reactive with DAN and related diazo reagents in the presence of cupric ions and the other reactive with EPNP, and that pepstatin binds in the vicinity of these residues. 2. The Type A acid protease from the same mold, on the other hand, was found to be markedly less sensitive to these specific inhibitors. Under conditions where the Type B enzyme was completely inactivated by DAN and related diazo reagents, only partial inactivation of this enzyme occurred. The effect of prior mixing of DAN and cupric ions on the pH profile of inactivation was also different from that for the Type B enzyme. Moreover, the Type A enzyme was not inactivated by EPNP. These results thus indicate that the nature of the active site of the Type A enzyme is rather different from that of the Type B enzyme and hence that the Type A enzyme belongs to a different class of acid proteases from the Type B enzyme.     

Cloning and nucleotide sequence of the highly thermostable neutral protease gene from Bacillus stearothermophilus

The gene (nprM) for the highly thermostable neutral protease of Bacillus stearothermophilus MK232 was cloned in Bacillus subtilis using pTB53 as a vector. The nucleotide sequence of nprM and its flanking regions was determined. The DNA sequence revealed only one large open reading frame, composed of 1656 base pairs and 552 amino acid residues. A Shine-Dalgarno (SD) sequence was found 12 bases upstream from the translation start site (ATG). A possible promotor sequence (TTTTCC for the -35 region and TATTGT for the -10 region), which was nearly identical to the promoter for another thermostable neutral protease gene, nprT, was also found about 40 bases upstream of the SD sequence. The deduced amino acid sequence contained a signal sequence in its amino-terminal region. The sequence of the first five amino acids of purified extracellular protease completely matched residues 237-241 of the open reading frame. This suggests that the enzyme is translated as a large polypeptide containing a pre-pro structure as is known for other neutral proteases. The amino acid sequence of the extracellular form of this protease (316 amino acids, molecular mass 34,266 Da) was identical to that of the thermostable neutral protease (thermolysin) from Bacillus thermoproteolyticus except for two amino acid substitutions (Asp37 to Asn37 and Glu119 to Gln119). The G + C content of the coding region of nprM was 42 mol%, while that of the third letter of the codons was lower (36 mol%). This extremely low content is an exceptional case for genes from thermophiles. When the protease genes, nprM and nprT, were cloned on pTB53 in B. subtilis, the expression of nprM was about 20 times higher than that of nprT. The reason for the difference between the two systems is discussed.     

Amino acid sequence of Salmonella typhimurium branched-chain amino acid aminotransferase

The complete amino acid sequence of the subunit of branched-chain amino acid aminotransferase (transaminase B, EC 2.6.1.42) of Salmonella typhimurium was determined. An Escherichia coli recombinant containing the ilvGEDAY gene cluster of Salmonella was used as the source of the hexameric enzyme. The peptide fragments used for sequencing were generated by treatment with trypsin, Staphylococcus aureus V8 protease, endoproteinase Lys-C, and cyanogen bromide. The enzyme subunit contains 308 residues and has a molecular weight of 33,920. To determine the coenzyme-binding site, the pyridoxal 5-phosphate containing enzyme was treated with tritiated sodium borohydride prior to trypsin digestion. Peptide map comparisons with an apoenzyme tryptic digest and monitoring radioactivity incorporation allowed identification of the pyridoxylated peptide, which was then isolated and sequenced. The coenzyme-binding site is the lysyl residue at position 159. The amino acid sequence of Salmonella transaminase B is 97.4% identical with that of Escherichia coli, differing in only eight amino acid positions. Sequence comparisons of transaminase B to other known aminotransferase sequences revealed limited sequence similarity (24-33%) when conserved amino acid substitutions are allowed and alignments were forced to occur on the coenzyme-binding site.     

Purification and amino acid sequence of a bitter gourd inhibitor against an acidic amino acid-specific endopeptidase of Streptomyces griseus.

An inhibitor (BGIA) against an acidic amino acid-specific endopeptidase of Streptomyces griseus (Glu S. griseus protease) was isolated from seeds of the bitter gourd Momordica charantia L., and its amino acid sequence was determined. The molecular weight of BGIA based on the amino acid sequence was calculated to be 7419. BGIA competitively inhibited Glu S. griseus protease with an inhibition constant (Ki) of 70 nM, and gel filtration analyses suggested that BGIA forms a 1:1 complex with this protease. However, two other acidic amino acid-specific endopeptidases, protease V8 from Staphylococcus aureus and Bacillus subtilis proteinase (Glu B. subtilis protease), were not inhibited by BGIA. BGIA had no inhibitory activity against chymotrypsin, trypsin, porcine pancreatic elastase, and papain, although subtilisin Carlsberg was strongly inhibited. The amino acid sequence of BGIA shows similarity to potato chymotrypsin inhibitor, barley subtilisin-chymotrypsin inhibitor CI-1 and CI-2, and leech eglin C, especially around the reactive site. Although the residue at the putative reactive site of these inhibitors is leucine or methionine, the corresponding amino acid in BGIA is alanine.     

The multiplicity of human pancreatic secretory trypsin inhibitor

Four forms of pancreatic secretory trypsin inhibitor (PSTI; A1, A2, B, and C) were purified from human pancreatic juice. According to sequence results, the primary structure of B was different from that reported earlier (Greene, L.J., et al. (1976) Method Enzymol. 45, 813-825) at two positions, i.e. Asn21----Asp21, Asp29----Asn29. A1 and A2 were deamidated forms of B judging from peptide mappings with Staphylococcus aureus V8 protease. Gln45 in B was replaced by Glu in A1 and Gln51 in B was replaced by Glu in A2. C was an inhibitor lacking five amino acid residues from the amino terminal of B. B and C inhibited human cationic trypsin activity stoichiometrically with similar dissociation constants, but A1 and A2 showed poorer trypsin inhibitory activity than B and C.     

Amino acid sequence of an alpha-delta-glycophorin hybrid. A structure reciprocal to Sta delta-alpha-glycophorin hybrid

In this report we examine the primary sequence of a variant glycophorin obtained from erythrocytes of an individual who exhibits an unusual MNSs blood group phenotype. We show that this protein is a hybrid molecule constructed from sequences of alpha- and delta-glycophorins (glycophorins A and B) in a alpha-delta arrangement. Serological typing revealed that the donor's phenotype was M+N+S+s+U+; yet his erythrocytes reacted with some but not all examples of anti-S antisera. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a variant glycophorin band, and immunoblotting and reaction with N-glycanase suggested that its amino terminus resembled that of M-alpha-glycophorin but that its carboxyl terminus did not. A preparation highly enriched in the variant was obtained and used to generate peptide fragments for sequencing. The sequence revealed that the variant was a hybrid molecule whose amino terminus corresponded to M-alpha-glycophorin and whose carboxyl terminus corresponded to S-delta-glycophorin. CNBr cleavage of the variant glycophorin yielded four peptides. The sequence of the amino-terminal CNBr peptide (residues 1-8) was identical to the amino-terminal octapeptide of M-alpha-glycophorin. The proceeding peptide (residues 9-61) contained a segment identical to residues 9-58 of alpha glycophorin, but its carboxyl-terminal sequence had the Gly-Glu-Met sequence from S-delta-glycophorin (residues 27-29). The other two peptides, insoluble in aqueous solutions, contained highly hydrophobic sequences, identical to residues 30-52 and 53-68 of delta-glycophorin. Sequences of overlapping peptides generated by trypsin and V8 protease confirmed the hybrid nature of the variant glycophorin: residues 1-58 were identical to residues 1-58 of M-alpha-glycophorin, and residues 59-100 were entirely identical to residues 27-68 of S-delta-glycophorin. The variant glycophorin is expected to have 4 additional residues at its carboxyl terminus that correspond to the carboxyl-terminal residues 69-72 of delta-glycophorin. The amino acid sequence arrangement of the variant alpha-delta-glycophorin is an exact reciprocal of that found in another hybrid glycophorin, Sta, that is a delta-alpha hybrid. We propose that the two hybrid glycophorins represent the two possible products resulting from a reciprocal recombination event.     

The primary structure of staphylococcal protease.

The amino acid sequence of staphylococcal protease has been determined by analysis of tryptic peptides obtained from cyanogen bromide fragments. Selected peptides obtained from digests with staphylococcal protease, thermolysin, and chymotrypsin provided the information necessary to align the tryptic peptides and the cyanogen bromide fragments. The protease is a single polypeptide chain of some 250 amino acids and is devoid of sulfhydryl groups. The COOH-terminal tryptic peptide of of the protease molecule contains some 43 residues, most of which are aspartic acids, asparagines, and prolines. The amino acid sequence of this peptide was not determined. The primary structure near the active serine residue indicates that staphylococcal protease is related to the pancreatic serine proteases. However, it has little or no additional sequence homologies with these enzymes except for the regions near histidine-50 and aspartic acid - 91. These regions have striking similarities with the corresponding regions of protease B and the trypsin-like enzyme of Streptomyces griseus.     

Intracellular serine protease of Bacillus subtilis: sequence homology with extracellular subtilisins.

Intracellular serine protease was isolated from stationary-grown Bacillus subtilis A-50 cells and purified to homogeneity. The molecular weight of the enzyme is 31,000 +/- 1,000, with an isoelectric point of 4.3. Its amino acid composition is characteristically enriched in glutamic acid content, differing from that of extra-cellular subtilisins. The enzyme is completely inhibited with phenylmethylsulfonyl fluoride and ethylenediaminetetraacetic acid. Intracellular protease possesses negligible activity towards bovine serum albumin and hemoglobin, but has 5- to 20-fold higher specific activity against p-nitroanilides of benzyloxycarbonyl tripeptides than subtilisin BPN'. Esterolytic activity of the enzyme is also higher than that of subtilisin BPN'. The enzyme is sequence homologous with secretory subtilisins throughout 50 determined NH2-terminal residues, indicating the presence of duplicated structural genes for serine proteases in the B. subtilis genome. The occurrence of two homologous genes in the cell might accelerate the evolution of serine protease not only by the loosening of selective constrainst, but also by creation of sequence variants by means of intragenic recombination. Three molecular forms of intracellular protease were found, two of them with NH2-terminal glutamic acid and one minor form, three residues longer, with asparagine as NH2 terminus. These data indicate the possible presence of an enzyme precursor proteolytically modified during cell growth.     

Identification of the sequence on NS4A required for enhanced cleavage of the NS5A/5B site by hepatitis C virus NS3 protease.

In addition to NS3 protease, the NS4A protein is required for efficient cleavage of the nonstructural protein region of the hepatitis C virus polyprotein. To investigate the function and the sequence of NS4A required for the enhancement of NS3 protease activity, we developed an in vitro NS3 protease assay system consisting of three purified viral elements: (i) a recombinant NS3 protease which was expressed in Escherichia coli as a maltose-binding protein-NS3 fusion protein (MBP-NS3), (ii) synthetic NS4A fragments, and (iii) a synthetic peptide substrate which mimics the NS5A/5B junction. We showed that the NS3 protease activity of MBP-NS3 was enhanced in a dose-dependent manner by 4A18-40, which is a peptide composed of amino acid residues 18 to 40 of NS4A. The optimal activity was observed at a 10-fold molar excess of 4A18-40 over MBP-NS3. The coefficient for proteolytic efficiency, kcat/Km, of NS3 protease was increased by about 40 times by the addition of a 10-fold molar excess of 4A18-40. Using a series of truncations of 4A18-40, we estimated that amino acid residues 22 to 31 in NS4A (SVVIVGRIIL) constituted the core sequence for the effector activity. Single-substitution experiments with 4A21-34, a peptide composed of amino acid residues 21 to 34 of NS4A, suggested the importance of several residues (Val-23, Ile-25, Gly-27, Arg-28, Ile-29, and Leu-31) for its activity. In addition, we found that some single-amino-acid substitutions in 4A21-34 were able to inhibit the enhancement of NS3 protease activity by 4A18-40. This approach has potential as a novel strategy for inhibiting the NS3 protease activity important for hepatitis C virus proliferation.     

Complete amino acid sequence of Scytalidium lignicolum acid protease B

The acid protease B (SLB) of Scytalidium lignicolum was reduced and carboxymethylated and then subjected to tryptic digestion. Five fragments were isolated and some of them were further digested with alpha-chymotrypsin, thermolysin, and dilute acetic acid. The sequence analysis of these fragments and the peptides by conventional methods established the complete amino acid sequence of SLB. The enzyme was composed of 204 amino acid residues with threonine and valine as its amino- and carboxyl-termini, respectively. Locations of three disulfide bridges were also established to be Cys47-126, Cys140-163, and Cys192-201 by enzymatic fragmentation of the denatured and unmodified SLB. Only a slight homology was found in the sequences of SLB and other acid proteases hitherto reported.     

Chemical modification of neutral protease from Bacillus subtilis var. amylosacchariticus with tetranitromethane: assignment of tyrosyl residues nitrated

A neutral protease from Bacillus subtilis var. amylosacchariticus was modified with tetranitromethane (TNM) at pH 8.0 for 1 h at 25 degrees C, by which treatment the proteolytic activity toward casein was markedly reduced, whereas activity changes toward N-blocked peptide substrates were variable depending upon the substrate used. The modified enzyme was digested with a Staphylococcus aureus V8 protease at pH 7.9 and the resultant peptides were separated by HPLC. Two peptides which contain nitrotyrosyl residue(s) were purified. One of the peptides was found to have an amino acid sequence of Thr-Ala-Asn-Leu-Ile-Tyr-Glu, which corresponds to residue Nos. 153-159 of the neutral protease, and Tyr-158 was identified as PTH-nitrotyrosine. The other one was the amino-terminal peptide of residue Nos. 1-22, and Tyr-21 was shown to be nitrated. From a comparison with the active site structure of thermolysin, which is a zinc metalloprotease with a high sequence homology to B. subtilis neutral proteases, nitration of Tyr-158 was inferred to be closely related to the activity changes of the neutral protease from B. subtilis var. amylosacchariticus.     

Identification of koningic acid (heptelidic acid)-modified site in rabbit muscle glyceraldehyde-3-phosphate dehydrogenase

The sesquiterpene antibiotic koningic acid (heptelidic acid) has been previously demonstrated to modify glyceraldehyde-3-phosphate dehydrogenase in specific manner, probably by binding to the sulfhydryl residue at the active site of the enzyme (Sakai, K., Hasumi, K. and Endo, A. (1988) Biochim. Biophys. Acta 952, 297-303). Rabbit muscle glyceraldehyde-3-phosphate dehydrogenase labeled with [3H]koningic acid was digested with trypsin. Reverse-phase HPLC revealed that the label is associated exclusively with a tryptic peptide having 17 amino acid residues. Microsequencing and fast atom bombardment mass spectrometry demonstrated that the peptide has the sequence Ile-Var-Ser-Asn-Ala-Ser-Cys-Thr-Thr-Asn-Cys-Leu-Ala-Pro-Leu-Ala-Lys. In comparison to the amino acid sequence of glyceraldehyde-3-phosphate dehydrogenase from other species, this peptide is in a highly conserved region and is part of the active site of the enzyme. The cysteine residue corresponding to the Cys-149 in the pig muscle enzyme, which has been shown to be an essential residue for the enzyme activity, was shown to be the site modified by koningic acid. Structural analyses of the reaction product of koningic acid and L-cysteine suggested that the epoxide of koningic acid reacts with the sulfhydryl group of cysteine residue, resulting in a thioether.     

Amino acid sequences of substrate-binding sites in chicken liver fatty acid synthase

The amino acid sequences of three essential regions of chicken liver fatty acid synthase have been determined: that around 4'-phosphopantetheine ("carrier" site), the substrate "loading" site containing serine, and a "waiting" site for the growing fatty acid containing cysteine. The amino acid sequence of the 4'-phosphopantetheine region was determined for the acetyl-, malonyl-, hydroxybutyryl-, and butyryl-enzyme with peptides obtained by hydrolysis of the enzyme with trypsin and Staphylococcus aureus (V8) protease. The sequence region around the essential serine was obtained for the acetyl- and malonyl-enzyme. The N-terminus of the tryptic peptide was blocked. However, the same sequence is obtained for the acetyl- and malonyl-peptide after S. aureus protease digestion, suggesting that the enzyme contains a single acyl transferase rather than two separate transacylases. The sequence around the cysteine was obtained by use of a radioactive iodoacetamide label. An unusual sequence of three serines adjacent to the cysteine was found. The strong similarities between peptides from different species for all three of the regions suggest that the multifunctional polypeptides from yeast and animals have evolved from the monofunctional enzymes of lower species.     

Reduced-bond tight-binding inhibitors of HIV-1 protease. Fine tuning of the enzyme subsite specificity.

Truncation of a peptide substrate in the N-terminus and replacement of its scissile amide bond with a non-cleavable reduced bond results in a potent inhibitor of HIV-1 protease. A series of such inhibitors has been synthesized, and S2-S3' subsites of the protease binding cleft mapped. The S2 pocket requires bulky Boc or PIV groups, large aromatic Phe residues are preferred in P1 and P1' and Glu in P2'. The S3' pocket prefers Phe over small Ala or Val. Introduction of a Glu residue into the P2' position yields a tight-binding inhibitor of HIV-1 protease, Boc-Phe-[CH2-NH]-Phe-Glu-Phe-OMe, with a subnanomolar inhibition constant. The relevant peptide derived from the same amino acid sequence binds to the protease with a Ki of 110 nM, thus still demonstrating a good fit of the amino acid residues into the protease binding pockets and also the importance of the flexibility of P1-P1' linkage for proper binding. A new type of peptide bond mimetic, N-hydroxylamine -CH2-N(OH)-, has been synthesized. Binding of hydroxylamino inhibitor of HIV-1 protease is further improved with respect to reduced-bond inhibitor.     

Streptococcal phosphoenolpyruvate-sugar phosphotransferase system: amino acid sequence and site of ATP-dependent phosphorylation of HPr

The amino acid sequence of histidine-containing protein (HPr) from Streptococcus faecalis has been determined by direct Edman degradation of intact HPr and by amino acid sequence analysis of tryptic peptides, V8 proteolytic peptides, thermolytic peptides, and cyanogen bromide cleavage products. HPr from S. faecalis was found to contain 89 amino acid residues, corresponding to a molecular weight of 9438. The amino acid sequence of HPr from S. faecalis shows extended homology to the primary structure of HPr proteins from other bacteria. Besides the phosphoenolpyruvate-dependent phosphorylation of a histidyl residue in HPr, catalyzed by enzyme I of the bacterial phosphotransferase system, HPr was also found to be phosphorylated at a seryl residue in an ATP-dependent protein kinase catalyzed reaction [Deutscher, J., & Saier, M. H., Jr. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 6790-6794]. The site of ATP-dependent phosphorylation in HPr of S. faecalis has now been determined. [32P]P-Ser-HPr was digested with three different proteases, and in each case, a single labeled peptide was isolated. Following digestion with subtilisin, we obtained a peptide with the sequence -(P)Ser-Ile-Met-. Using chymotrypsin, we isolated a peptide with the sequence -Ser-Val-Asn-Leu-Lys-(P)Ser-Ile-Met-Gly-Val-Met-. The longest labeled peptide was obtained with V8 staphylococcal protease. According to amino acid analysis, this peptide contained 36 out of the 89 amino acid residues of HPr. The following sequence of 12 amino acid residues of the V8 peptide was determined: -Tyr-Lys-Gly-Lys-Ser-Val-Asn-Leu-Lys-(P)Ser-Ile-Met-.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cloning and characterization of Thermotoga maritima beta-glucuronidase

The putative beta-glucuronidase from Thermotoga maritima, comprising 563 amino acid residues conjugated with a Hisx6 tag, was cloned and expressed in Escherichia coli. The enzyme has a moderately broad specificity, hydrolysing a range of p-nitrophenyl glycoside substrates, but has greatest activity on p-nitrophenyl beta-D-glucosiduronic acid (kcat=68 s(-1), kcat/K(M)= 4.5x10(5) M(-1) s(-1)). The enzyme also shows a relatively broad pH-dependence with activity from pH4.5 to 7.5 and a maximum at pH6.5. As expected the enzyme is stable towards heat denaturation, with a half life of 3h at 85 degrees C, in contrast to the mesophilic E. coli enzyme, which has a half life of 2.6h at 50 degrees C. The identity of the catalytic nucleophile was confirmed as Glu476 within the sequence VTEFGAD by trapping the glycosyl-enzyme intermediate using the mechanism-based inactivator, 2-deoxy-2-fluoro-beta-D-glucosyluronic acid fluoride and identifying the labeled peptide in peptic digests by HPLC-MS/MS methodologies. Consistent with this, the Glu476Ala mutant was shown to be hydrolytically inactive. The acid/base catalyst was confirmed as Glu383 by generation and kinetic analysis of enzyme mutants modified at that position, Glu383Ala and Glu383Gln. The demonstration of activity rescue by azide is consistent with the proposed role for this residue. This enzyme therefore appears suitable for use in enzymatic oligosaccharide synthesis in either the transglycosylation mode or by use of glycosynthase and thioglycoligase approaches.