Identification of Asp-130 as the catalytic nucleophile in the main alpha-galactosidase from Phanerochaete chrysosporium, a family 27 glycosyl hydrolase

Characterization of the complete gene sequence encoding the alpha-galactosidase from Phanerochaete chrysosporium confirms that this enzyme is a member of glycosyl hydrolase family 27 [Henrissat, B., and Bairoch, A. (1996) Biochem. J. 316, 695-696]. This family, together with the family 36 alpha-galactosidases, forms glycosyl hydrolase clan GH-D, a superfamily of alpha-galactosidases, alpha-N-acetylgalactosaminidases, and isomaltodextranases which are likely to share a common catalytic mechanism and structural topology. Identification of the active site catalytic nucleophile was achieved by labeling with the mechanism-based inactivator 2',4', 6'-trinitrophenyl 2-deoxy-2,2-difluoro-alpha-D-lyxo-hexopyranoside; this inactivator was synthesized by anomeric deprotection of the known 1,3,4,6-tetra-O-acetyl-2-deoxy-2, 2-difluoro-D-lyxo-hexopyranoside [McCarter, J. D., Adam, M. J., Braun, C., Namchuk, M., Tull, D., and Withers, S. G. (1993) Carbohydr. Res. 249, 77-90], picrylation with picryl fluoride and 2, 6-di-tert-butylpyridine, and O-deacetylation with methanolic HCl. Enzyme inactivation is a result of the formation of a stable 2-deoxy-2,2-difluoro-beta-D-lyxo-hexopyranosyl-enzyme intermediate. Following peptic digestion, comparative liquid chromatographic/mass spectrometric analysis of inactivated and control enzyme samples served to identify the covalently modified peptide. After purification of the labeled peptide, benzylamine was shown to successfully replace the 2-deoxy-2,2-difluoro-D-lyxo-hexopyranosyl peptidyl ester by aminolysis. The labeled amino acid was identified as Asp-130 of the mature protein by further tandem mass spectrometric analysis of the native and derivatized peptides in combination with Edman degradation analysis. Asp-130 is found within the sequence YLKYDNC, which is highly conserved in all known family 27 glycosyl hydrolases.     

Reaction of substrates with 35S-thiophosphorylated succinyl-CoA synthetase of pig heart. Similarities to the case of the Escherichia coli enzyme

Guanosine 5'-O-(3-thio)triphosphate (GTP gamma S) was found to be a substrate of pig heart succinyl-CoA synthetase with Km and kcat values of 3 microM and 0.23 s-1, respectively. The corresponding values with GTP as substrate were 48 microM and 65 s-1. 35S-thiophosphorylated enzyme was prepared by incubation of pig heart succinyl-CoA synthetase with [35S]GTP gamma S. A comparison was made of thiophosphoryl group release by substrates from this alpha beta (one active site) enzyme with that of the alpha 2 beta 2 (two active sites) Escherichia coli enzyme (Wolodko, W. T., Brownie, E. R., O'Connor, M. D., and Bridger, W. A. (1983) J. Biol. Chem. 258, 14116-14119; Nishimura, J. S., and Mitchell, T. (1984) J. Biol. Chem. 259, 9642-9645). It was found, as in the case of the E. coli enzyme, that thiophosphoryl group release by GDP and by succinate plus CoA was stimulated by succinyl-CoA and GTP, respectively. The same result was observed at 1, 0.1, and 0.01 mg/ml, lending assurance that these phenomena were not exhibited by an aggregated form of the pig heart enzyme. While an alternating-sites catalytic cooperativity model is not ruled out for the E. coli enzyme, it is proposed that the NTP- and succinyl-CoA-stimulated release of thiophosphoryl groups from either enzyme involves a "same-site" mechanism, to be distinguished from an "other-site" mechanism.     

Processing of the papain precursor. Purification of the zymogen and characterization of its mechanism of processing

The precursor of the cysteine protease papain has been expressed and secreted as propapain from insect cells infected with a recombinant baculovirus expressing a synthetic gene coding for prepropapain. This 39-kDa secreted propapain zymogen molecule is glycosylated and can be processed in vitro into an enzymatically active authentic papain molecule of 24.5 kDa (Vernet, T., Tessier, D.C., Richardson, C., Laliberté, F., Khouri, H. E., Bell, A. W., Storer, A. C., and Thomas, D. Y. (1990) J. Biol. Chem. 265, 16661-16666). Recombinant propapain was stabilized with Hg2+ and purified to homogeneity using affinity chromatography, gel filtration, and ion-exchange chromatographic procedures. The maximum rate of processing in vitro was achieved at approximately pH 4.0, at a temperature of 65 degrees C and under reducing conditions. Precursor processing is inhibited by a variety of reversible and irreversible cysteine protease inhibitors but not by specific inhibitors of serine, metallo or acid proteases. Replacement by site-directed mutagenesis of the active site cysteine with a serine at position 25 also prevents processing. The inhibitor 125I-N-(2S,3S)-3-trans-hydroxycarbonyloxiran-2-carbonyl-L-tyrosine benzyl ester covalently labeled the wild type papain precursor, but not the C25S mutant, indicating that the active site is accessible to the inhibitor and is in a native conformation within the precursor. Based on biochemical and kinetic analyses of the activation and processing of propapain we have shown that the papain precursor is capable of autoproteolytic cleavage (intramolecular). Once free papain is released processing can then occur in trans (intermolecular).     

Spectral evidence for tyrosine ionization linked to a conformational change in liver alcohol dehydrogenase ternary complexes.

Resonance energy transfer from Trp-314 to ionized Tyr-286 was proposed (Laws, W. R., and Shore, J. D. (1978) J. Biol. Chem. 253, 8593-8597) as the mechanism for the observed decrease in protein fluorescence of liver alcohol dehydrogenase seen with alkaline pH, or with the formation of a ternary complex with NAD+ and trifluoroethanol. In the present study, ultraviolet difference spectra confirm the presence of ionized tyrosine not only in these two cases but also in the ternary complex with NADH and isobutyramide. Our results indicate that ternary complex formation, with either oxidized or reduced coenzyme, causes a conformational change leading to partial ionization of tyrosine residues in regions of the enzyme far from the active site.     

A structural model for across membrane coupling between the Qo and Qi active sites of cytochrome bc1

The two spatially distant quinone-binding sites of the ubihydroquinone: cytochrome c oxidoreductase (cyt bc₁) complex have been shown to influence one another in some fashion. This transmembrane communication alters cofactor and redox partner binding interactions and could potentially influence the timing or ‘concerted’ steps involved in the steady-state turnover of the homodimeric enzymes. Yet, despite several lines of evidence corroborating the coupling of the quinone binding active sites to one another, little to no testable hypothesis has been offered to explain how such a “signal” might be transmitted across the presumably rigid hydrophobic domain of the enzyme. Recently, it has been shown that this interquinone binding sites communication influences the steady-state position of the mobile [2Fe–2S] cluster containing iron sulfur protein (Sarewicz M., Dutka M., Froncisz W., Osyczka A. (2009) Biochemistry 48, 5708–5720) as mediated by at least one transmembrane helix of the b-type cyt containing subunit (Cooley, J. W., Lee, D. W., and Daldal, F. (2009) Biochemistry 48, 1988–1999). Here we provide an overview of the evidence supporting the structural coupling of these sites and provide a theoretical framework for how the redox state of a quinone at one cofactor binding site might influence the cofactor–, inhibitor–, and/or protein–protein interactions at the structurally distant opposing Q binding site.     

Reaction of Lactobacillus histidine decarboxylase with L-histidine methyl ester

L-Histidine methyl ester inactivates histidine decarboxylase in a time-dependent manner. The possibility was considered that an irreversible reaction between enzyme and inhibitor occurs [Recsei, P. A., & Snell, E. E. (1970) Biochemistry 9, 1492-1497]. We have confirmed time-dependent inactivation by histidine methyl ester and have investigated the structure of the enzyme-inhibitor complex. Upon exposure to either 8 M guanidinium chloride or 6% trichloroacetic acid, unchanged histidine methyl ester is recovered. Formation of the complex involves Schiff base formation, most likely with the active site pyruvyl residue [Huynh, Q. K., & Snell, E. E. (1986) J. Biol. Chem. 261, 4389-4394], but does not involve additional irreversible covalent interaction between inhibitor and enzyme. Complex formation is a two-step process involving rapidly reversible formation of a loose complex and essentially irreversible formation of a tight complex. For the formation of the tight complex, Ki = 80 nM and koff = 2.5 X 10(-4) min-1. Time-dependent inhibition was also observed with L-histidine ethyl ester, L-histidinamide, and DL-3-amino-4-(4-imidazolyl)-2-butanone. No inactivation was observed with glycine methyl ester or histamine. We propose that in the catalytic reaction the carboxyl group of the substrate is in a hydrophobic region. The unfavorable interaction between the carboxylate group and the hydrophobic region facilitates decarboxylation [Crosby, J., Stone, R., & Liehard, G. E. (1970) J. Am. Chem. Soc. 92, 2891-2900]. With histidine methyl ester this unfavorable interaction is no longer present; hence, there is tight binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanistic studies of substrate-assisted inhibition of ubiquitin-activating enzyme by adenosine sulfamate analogues

Ubiquitin-activating enzyme (UAE or E1) activates ubiquitin via an adenylate intermediate and catalyzes its transfer to a ubiquitin-conjugating enzyme (E2). MLN4924 is an adenosine sulfamate analogue that was identified as a selective, mechanism-based inhibitor of NEDD8-activating enzyme (NAE), another E1 enzyme, by forming a NEDD8-MLN4924 adduct that tightly binds at the active site of NAE, a novel mechanism termed substrate-assisted inhibition (Brownell, J. E., Sintchak, M. D., Gavin, J. M., Liao, H., Bruzzese, F. J., Bump, N. J., Soucy, T. A., Milhollen, M. A., Yang, X., Burkhardt, A. L., Ma, J., Loke, H. K., Lingaraj, T., Wu, D., Hamman, K. B., Spelman, J. J., Cullis, C. A., Langston, S. P., Vyskocil, S., Sells, T. B., Mallender, W. D., Visiers, I., Li, P., Claiborne, C. F., Rolfe, M., Bolen, J. B., and Dick, L. R. (2010) Mol. Cell 37, 102-111). In the present study, substrate-assisted inhibition of human UAE (Ube1) by another adenosine sulfamate analogue, 5'-O-sulfamoyl-N(6)-[(1S)-2,3-dihydro-1H-inden-1-yl]-adenosine (Compound I), a nonselective E1 inhibitor, was characterized. Compound I inhibited UAE-dependent ATP-PP(i) exchange activity, caused loss of UAE thioester, and inhibited E1-E2 transthiolation in a dose-dependent manner. Mechanistic studies on Compound I and its purified ubiquitin adduct demonstrate that the proposed substrate-assisted inhibition via covalent adduct formation is entirely consistent with the three-step ubiquitin activation process and that the adduct is formed via nucleophilic attack of UAE thioester by the sulfamate group of Compound I after completion of step 2. Kinetic and affinity analysis of Compound I, MLN4924, and their purified ubiquitin adducts suggest that both the rate of adduct formation and the affinity between the adduct and E1 contribute to the overall potency. Because all E1s are thought to use a similar mechanism to activate their cognate ubiquitin-like proteins, the substrate-assisted inhibition by adenosine sulfamate analogues represents a promising strategy to develop potent and selective E1 inhibitors that can modulate diverse biological pathways.     

The lysine-49 phospholipase A2 from the venom of Agkistrodon piscivorus piscivorus. Relation of structure and function to other phospholipases A2

A new class of phospholipases A2 that have a lysine at position 49 differ from the more conventional Asp-49 enzymes with respect to the sequential binding of the essential cofactor, calcium, and the substrate, phospholipid, in the formation of the catalytic complex (Maraganore, J.M., Merutka, G., Cho, W., Welches, W., Kézdy, F.J., and Heinrikson, R.L. (1984) J. Biol. Chem. 259, 13839-13843). We report here the complete amino acid sequence of the Lys-49 enzyme from Agkistrodon piscivorus piscivorus. The sequence was determined by automated Edman degradation of the intact, S-carboxymethylcysteinyl protein and of peptides derived therefrom by cleavage with cyanogen bromide, chymotrypsin, trypsin, and endoproteinase Lys-C. Despite several changes at amino acid residues previously considered to be invariant, the Lys-49 enzymes are homologous to the Asp-49 phospholipases. Homology is especially apparent in the following: 1) the pattern of 14 half-cystine residues, 2) conservation of hydrophobic residues which have been shown to encircle the active site, and 3) conservation of Asp-99 and His-48 which have been implicated in the catalytic reaction itself. These observations together with kinetic and binding data imply that the Lys-49 phospholipases have a catalytic mechanism and a three-dimensional architecture similar to those of the Asp-49 enzymes. Modeling of the Lys-49 enzyme based upon the structure of bovine pancreatic phospholipase reveals that the epsilon-amino group of Lys-49 can fit easily in the calcium-binding site and, moreover, that this orientation of a cationic side chain at position 49 could account for the characteristic and novel feature of the Lys-49 phospholipases, i.e. that they are able to form complexes with phospholipid in the absence of calcium.     

Kinetic evidence that the high-affinity glucose 6-phosphate site on hexokinase I Is the active site

Two mechanisms have been suggested to account for the regulation of brain hexokinase by glucose 6-phosphate. One mechanism places glucose-6-P at an allosteric site, remote from the active site, while the second describes glucose-6-P as a simple product inhibitor of the enzyme, binding at the γ phosphate subsite within the ATP locus of the active site. To distinguish between these possibilities, we have undertaken a study of the back reaction of hexokinase I. Our data indicate that glucose-6-P displays classical Michaelis-Menten kinetics with brain hexokinase. This finding is consistent only with the high-affinity glucose-6-P site on the enzyme being the catalytic site. The dissociation constant, estimated from the initial-rate experiments is approximately 25 μm, a value that agrees well with the inhibition constant for glucose-6-P in the forward direction. These findings are consistent with an earlier model (W. R. Ellison, J. D. Lueck and H. J. Fromm, (1975) J. Biol. Chem.250, 1864–1871), which maintains that glucose-6-P inhibition of brain hexokinase is a manifestation of product inhibition. In a recent paper, Lazo et al. (P. A. Lazo, A. Sols, and J. E. Wilson, (1980) J. Biol. Chem.255, 7548–7551) reported data obtained from binding studies with rat brain hexokinase at an elevated (250 μm) level of glucose-6-P. These authors believe that their results indicate multiple binding of glucose-6-P to the enzyme and interpret the data in terms of a high-affinity allosteric site and a low-affinity catalytic site. Our results are at variance with this interpretation and are consistent only with the high-affinity site for glucose-6-P on brain hexokinase being the active site.     

X-ray diffraction structure of a plant glycosyl hydrolase family 32 protein: fructan 1-exohydrolase IIa of Cichorium intybus

Fructan 1-exohydrolase, an enzyme involved in fructan degradation, belongs to the glycosyl hydrolase family 32. The structure of isoenzyme 1-FEH IIa from Cichorium intybus is described at a resolution of 2.35 A. The structure consists of an N-terminal fivefold beta-propeller domain connected to two C-terminal beta-sheets. The putative active site is located entirely in the beta-propeller domain and is formed by amino acids which are highly conserved within glycosyl hydrolase family 32. The fructan-binding site is thought to be in the cleft formed between the two domains. The 1-FEH IIa structure is compared with the structures of two homologous but functionally different enzymes: a levansucrase from Bacillus subtilis (glycosyl hydrolase family 68) and an invertase from Thermotoga maritima (glycosyl hydrolase family 32).     

Studies of bovine liver glutamate dehydrogenase by analytical affinity chromatography on immobilized AMP analogs.

Bovine liver glutamate dehydrogenase has been studied by analytical affinity chromatography on two immobilized AMP analogs, i.e., N⁶-(6-aminohexyl)-AMP and 8-(6-aminohexyl)-amino-AMP. The existence of various enzyme-coenzyme and enzyme-effector complexes has been verified. Also the cooperative formation of two ternary complexes, i.e., glutamic dehydrogenase (GHD)-NADP-glutamate and GDH-ADP-leucine, has been shown. The results of this study have been rationalized by the “ligand exclusion theory.” which has been proposed for the regulation of the glutamic dehydrogenase. It has been shown that the active site and the ADP-binding effector site are oriented close to each other on the enzyme. Furthermore, the data suggest that the adenylic site is not identical to the nonactive coenzyme binding site. A mechanism based on electrostatic interactions is suggested for the cooperative binding of oxidized coenzyme and substrate. Dissociation constants for complexes between the enzyme and two coenzyme fragments (P-ADPR and 2′,5′-ADP) have been estimated.     

Kinetics and mechanism of refolding of bovine carbonic anhydrase. A probe study of the formation of the active site.

In kinetic studies of the folding of bovine carbonic anhydrase from disorganized to native structure, an azosulfonamide, 2-(4-sulfomylphenylazo)-7-acetamido-1-hydroxynaphthalene-3,6-disulfonate (I), has been used as a probe to follow the dynamics of formation of the active site region. The probe is a specific inhibitor of the native enzyme that binds in the active site crevice. The experiments, with previous data (Yazgan, A., and Henkens, R. W. (1972), Biochemistry 11, 1314), show that a tight binding site for I forms at an intermediate stage in the folding process. A subsequent conformational change perturbs the visible absorption and circular dichroism of bound I and could result in even tighter binding. The subsequent change completes formation of the active site. This is shown by results from separate experiments on the kinetics of recovery of activity (p-nitrophenyl acetate as substrate). Similar probe methods could be used with other proteins and enzymes to study the kinetics and mechanism of regeneration of specific sites--for example, the active site.     

The heparin/heparan sulfate 2-O-sulfatase from Flavobacterium heparinum. A structural and biochemical study of the enzyme active site and saccharide substrate specificity

In the previous paper (Myette, J. R., Shriver, Z., Claycamp, C., McLean, M. W., Venkataraman, G., and Sasisekharan, R. (2003) J. Biol. Chem. 278, 12157-12166), we described the molecular cloning, recombinant expression, and preliminary biochemical characterization of the heparin/heparan sulfate 2-O-sulfatase from Flavobacterium heparinum. In this paper, we extend our structure-function investigation of the 2-O-sulfatase. First, we have constructed a homology-based structural model of the enzyme active site, using as a framework the available crystallographic data for three highly related arylsulfatases. In this model, we have identified important structural parameters within the enzyme active site relevant to enzyme function, especially as they relate to its substrate specificity. By docking various disaccharide substrates, we identified potential structural determinants present within these substrates that would complement this unique active site architecture. These determinants included the position and number of sulfates present on the glucosamine, oligosaccharide chain length, the presence of a Delta4,5-unsaturated double bond, and the exolytic versus endolytic potential of the enzyme. The predictions made from our model provided a structural basis of substrate specificity originally interpreted from the biochemical and kinetic data. Our modeling approach was further complemented experimentally using peptide mapping in tandem with mass spectrometry and site-directed mutagenesis to physically demonstrate the presence of a covalently modified cysteine (formylglycine) within the active site. This combinatorial approach of structure modeling and biochemical studies provides insight into the molecular basis of enzyme function.     

Competitive inhibitors of Klebsiella aerogenes urease. Mechanisms of interaction with the nickel active site

We examined several compounds for their mechanisms of inhibition with the nickel-containing active site of homogeneous Klebsiella aerogenes urease. Thiolate anions competitively inhibit urease and directly interact with the metallocenter, as shown by the pH dependence of inhibition and by UV-visible absorbance spectroscopic studies. Cysteamine, which possesses a cationic beta-amino group, exhibited a high affinity for urease (Ki = 5 microM), whereas thiolates containing anionic carboxyl groups were uniformly poor inhibitors. Phosphate monoanion competitively inhibits a protonated form of urease with a pKa of less than 5. Both the thiolate and phosphate inhibition results are consistent with charge repulsion by an anionic group in the urease active site. Acetohydroxamic acid (AHA) was shown to be a slow-binding competitive inhibitor of urease. This compound forms an initial E.AHA complex which then undergoes a slow transformation to yield an E.AHA* complex; the overall dissociation constant of AHA is 2.6 microM. Phenylphosphorodiamidate, also shown to be a slow-binding competitive inhibitor, possesses an overall dissociation constant of 94 pM. The tight binding of phenylphosphorodiamidate was exploited to demonstrate the presence of two active sites per enzyme molecule. Urease contains 4 mol of nickel/mol enzyme, hence there are two nickel ions/catalytic unit. Each of the two slow-binding inhibitors are proposed to form complexes in which the inhibitor bridges the two active site nickel ions. The inhibition results obtained for K. aerogenes urease are compared with inhibition studies of other ureases and are interpreted in terms of a model for catalysis proposed for the jack bean enzyme (Dixon, N.E., Riddles, P.W., Gazzola, C., Blakely, R.L., and Zerner, B. (1980) Can. J. Biochem. 58, 1335-1344).     

Glycosyl fluorides in enzymatic reactions

Glycosyl fluorides have considerable importance as substrates and inhibitors in enzymatic reactions. Their good combination of stability and reactivity has enabled their use as glycosyl donors with a variety of carbohydrate processing enzymes. Moreover, the installation of fluorine elsewhere on the carbohydrate scaffold commonly modifies the properties of the glycosyl fluoride such that the resultant compounds act as slow substrates or even inhibitors of enzyme action. This review covers the use of glycosyl fluorides as substrates for wild-type and mutant glycosidases and other enzymes that catalyze glycosyl transfer. The use of substituted glycosyl fluorides as inhibitors of enzymes that catalyze glycosyl transfer and as tools for investigation of their mechanism is discussed, including the labeling of active site residues. Synthetic applications in which glycosyl fluorides are used as glycosyl donors in enzymatic transglycosylation reactions for the synthesis of oligo- and polysaccharides are then covered, including the use of mutant glycosidases, the so-called glycosynthases, which are able to catalyze the formation of glycosides without competing hydrolysis. Finally, a short overview of the use of glycosyl fluorides as substrates and inhibitors of phosphorylases and phosphoglucomutase is given.     

Kinase FA-mediated regulation of rabbit skeletal muscle protein phosphatase. Reversible phosphorylation of the modulator subunit

A mechanism of activation of the ATP.Mg-dependent protein phosphatase (FC.M) has been proposed (Jurgensen, S., Shacter, E., Huang, C. Y., Chock, P. B., Yang, S.-D., Vandenheede, J. R., and Merlevede, W. (1984) J. Biol. Chem. 259, 5864-5870) in which a transient phosphorylation by the kinase FA of the modulator subunit (M) is the driving force for the transition of the inactive catalytic subunit (FC) into its active conformation. Incubation of FC.M with kinase FA and Mg2+ and adenosine 5'-(gamma-thio)triphosphate results in thiophosphorylation of M and also a conformational change in the phosphatase catalytic subunit; however, the enzyme remains inactive. Proteolysis of this inactive, thiophosphorylated complex causes proteolytic destruction of the modulator subunit and yields an active phosphorylase phosphatase species. Similar treatment of the native inactive enzyme does not yield active phosphatase. Evidence is presented, suggesting that a molecule of modulator is bound at an "inhibitory site" on the native enzyme. This modulator does not prevent the conformational change in the phosphatase catalytic subunit upon incubation with kinase FA and ATP.Mg but does partially inhibit the expression of the phosphorylase phosphatase activity.     

Two functional domains of coenzyme A activate catalysis by coenzyme A transferase. Pantetheine and adenosine 3'-phosphate 5'-diphosphate

Studies of the reactivity of succinyl-CoA:3-keto acid CoA transferase with a small coenzyme A analog, methylmercaptopropionate, have shown that noncovalent interactions between the enzyme and the side chain of CoA are responsible for a rate acceleration of approximately 10(12), which is close to the total rate acceleration brought about by the enzyme (Moore, S. A., and Jencks, W. P. (1982) J. Biol. Chem. 257, 10893-10907). We report here that interaction between the enzyme and the pantetheine moiety of CoA provides the majority of the rate acceleration and destabilization of the enzyme-thiol ester intermediate that is observed with CoA substrates. The role of the adenosine 3'-phosphate 5'-diphosphate moiety of CoA is to provide 6.9 kcal/mol of binding energy in order to pull the pantetheine moiety into the active site. The enzyme-thiol ester intermediate, E-pantetheine, was generated by reaction of pantetheine with the thiol ester of enzyme and methylmercaptopropionate. E-Pantetheine undergoes hydrolysis with khyd = 2 min-1, 140-fold faster than E-CoA, and reacts with acetoacetate with kAcAc = 3 X 10(6) M-1 min-1, only 10-fold slower than E-CoA. However, in the reverse direction acetoacetylpantetheine reacts with CoA transferase (kAcAc-SP = 220 M-1 min-1) 1.6 X 10(6) times slower than acetoacetyl-CoA. The equilibrium constant for the reaction of pantetheine with E-CoA is approximately 8 X 10(-6).     

Trimethoprim binds in a bacterial mode to the wild-type and E30D mutant of mouse dihydrofolate reductase

Previous crystallographic studies of the antibacterial trimethoprim in complexes with bacterial and avian dihydrofolate reductases have shown substantial differences in the mode of binding, providing plausible explanations for the origin of the remarkable species selectivity of this inhibitor (Matthews, D. A., Bolin, J. T., Burridge, J. M., Filman, D. J., Volz, K. W., Kaufman, B. T., Beddell, C. R., Champness, J. N., Stammers, D. K., and Kraut, J. (1985) J. Biol. Chem. 260, 381-391; Matthews, D. A., Bolin, J. T., Burridge, J. M., Filman, D. J., Volz, K. W., and Kraut, J. (1985) J. Biol. Chem. 260, 392-399). A major species difference between the active sites is that the only carboxylate present is always Glu in vertebrates and Asp in bacteria. Crystallographic studies of the wild-type and E30D mutant of the enzyme from mouse now reveal that in both cases trimethoprim is bound in an identical fashion to that observed with the bacterial enzyme, and there is no obvious single explanation for the origin of the 10(5)-fold selectivity of trimethoprim binding. In an earlier study of a mouse wild-type enzyme using more limited data it was proposed that trimethoprim bound in the avian mode (Stammers, D. K., Champness, J. N., Beddell, C. R., Dann, J. G., Eliopoulos, E. E., Geddes, A. J., Ogg, D., and North, A. C. T. (1987) FEBS Lett. 218, 178-184), but a re-examination indicates that the occupancy of the active site by trimethoprim is less than had been thought, and we are currently unable to make an unambiguous interpretation of the electron density maps and cannot confirm the avian mode of binding in those crystals.     

Crystal structure of a bacterial signal peptidase apoenzyme: implications for signal peptide binding and the Ser-Lys dyad mechanism.

We report here the x-ray crystal structure of a soluble catalytically active fragment of the Escherichia coli type I signal peptidase (SPase-(Delta2-75)) in the absence of inhibitor or substrate (apoenzyme). The structure was solved by molecular replacement and refined to 2.4 A resolution in a different space group (P4(1)2(1)2) from that of the previously published acyl-enzyme inhibitor-bound structure (P2(1)2(1)2) (Paetzel, M., Dalbey, R.E., and Strynadka, N.C.J. (1998) Nature 396, 186-190). A comparison with the acyl-enzyme structure shows significant side-chain and main-chain differences in the binding site and active site regions, which result in a smaller S1 binding pocket in the apoenzyme. The apoenzyme structure is consistent with SPase utilizing an unusual oxyanion hole containing one side-chain hydroxyl hydrogen (Ser-88 OgammaH) and one main-chain amide hydrogen (Ser-90 NH). Analysis of the apoenzyme active site reveals a potential deacylating water that was displaced by the inhibitor. It has been proposed that SPase utilizes a Ser-Lys dyad mechanism in the cleavage reaction. A similar mechanism has been proposed for the LexA family of proteases. A structural comparison of SPase and members of the LexA family of proteases reveals a difference in the side-chain orientation for the general base lysine, both of which are stabilized by an adjacent hydroxyl group. To gain insight into how signal peptidase recognizes its substrates, we have modeled a signal peptide into the binding site of SPase. The model is built based on the recently solved crystal structure of the analogous enzyme LexA (Luo, Y., Pfuetzner, R. A., Mosimann, S., Paetzel, M., Frey, E. A., Cherney, M., Kim, B., Little, J. W., and Strynadka, N. C. J. (2001) Cell 106, 1-10) with its bound cleavage site region.