Catalytic Carboxylate Research Articles

Crystal structures of Giardia lamblia guanine phosphoribosyltransferase at 1.75 A(,).

Giardia lamblia, the protozoan parasite responsible for giardiasis, requires purine salvage from its host for RNA and DNA synthesis. G. lamblia expresses an unusual purine phosphoribosyltransferase with a high specificity for guanine (GPRTase). The enzyme's sequence significantly diverges from those of related enzymes in other organisms. The transition state analogue immucillinGP is a powerful inhibitor of HGXPRTase from malaria [Li, C. M., et al. (1999) Nat. Struct. Biol. 6, 582-587] and is also a 10 nM inhibitor of G. lamblia GPRTase. Cocrystallization of GPRTase with immucillinGP led unexpectedly to a GPRTase.immucillinG binary complex with an open catalytic site loop. Diffusion of ligands into preformed crystals gave a GPRTase.immucillinGP.Mg(2+).pyrophosphate complex in which the open loop is stabilized by crystal contacts. G. lamblia GPRTase exhibits substantial structural differences from known purine phosphoribosyltransferases at positions remote from the catalytic site, but conserves most contacts to the bound inhibitor. The filled catalytic site with an open catalytic loop provides insight into ligand binding. One active site Mg(2+) ion is chelated to pyrophosphate, but the other is chelated to two conserved catalytic site carboxylates, suggesting a role for these amino acids. This arrangement of Mg(2+) and pyrophosphate has not been reported in purine phosphoribosyltransferases. ImmucillinG in the binary complex is anchored by its 9-deazaguanine group, and the iminoribitol is disordered. No Mg(2+) or pyrophosphate is detected; thus, the 5'-phosphoryl group is needed to immobilize the iminoribitol prior to magnesium pyrophosphate binding. Filling the catalytic site involves (1) binding the purine ring, (2) anchoring the 5'-phosphate to fix the ribosyl group, (3) binding the first Mg(2+) to Asp125 and Glu126 carboxyl groups and binding Mg(2+).pyrophosphate, and (4) closing the catalytic site loop and formation of bound (Mg(2+))(2). pyrophosphate prior to catalysis. Guanine specificity is provided by two peptide carbonyl oxygens hydrogen-bonded to the exocyclic amino group and a weak interaction to O6. Transition state formation involves N7 protonation by Asp129 acting as the general acid.

Relocation of the catalytic carboxylate group in class A beta-lactamase: the structure and function of the mutant enzyme Glu166-->Gln:Asn170-->Asp.

The hydrolysis of beta-lactam antibiotics by the serine-beta-lactamases proceeds via an acyl-enzyme intermediate. In the class A enzymes, a key catalytic residue, Glu166, activates a water molecule for nucleophilic attack on the acyl-enzyme intermediate. The active site architecture raises the possibility that the location of the catalytic carboxylate group may be shifted while still maintaining close proximity to the hydrolytic water molecule. A double mutant of the Staphylococcus aureus PC1 beta-lactamase, E166Q:N170D, was produced, with the carboxylate group shifted to position 170 of the polypeptide chain. A mutant protein, E166Q, without a carboxylate group and with abolished deacylation, was produced as a control. The kinetics of the two mutant proteins have been analyzed and the crystal structure of the double mutant protein has been determined. The kinetic data confirmed that deacylation was restored in E166Q:N170D beta-lactamase, albeit not to the level of the wild-type enzyme. In addition, the kinetics of the double mutant enzyme follows progressive inactivation, characterized by initial fast rates and final slower rates. The addition of ammonium sulfate increases the size of the initial burst, consistent with stabilization of the active form of the enzyme by salt. The crystal structure reveals that the overall fold of the E166Q:N170D enzyme is similar to that of native beta-lactamase. However, high crystallographic temperature factors are associated with the ohm-loop region and some of the side chains, including Asp170, are partially or completely disordered. The structure provides a rationale for the progressive inactivation of the Asp170-containing mutant, suggesting that the flexible ohm-loop may be readily perturbed by the substrate such that Asp170's carboxylate group is not always poised to facilitate hydrolysis.

Catalytic Thiol and Carboxylate: Role of Cysteine and Glutamic Acid in the Xylosidic Activity of Endoxylanase fromChainiasp. (NCL 82-5-1)

Chemical modification of the endoxylanase fromChainiasp. with group-specific chemical modifiers in the absence and presence of substrate and kinetics of modification revealed the involvement of a thiol and a carboxylate in the catalytic function of the enzyme. The active-site peptides were chemically labeled and sequenced. The sequence alignment of the chemically labeled peptide with other family G/11 xylanases showed that the catalytic glutamate ofChainiaxylanase is located in a highly homologous region and may function as an acid/base catalyst while thiol of the Cys may function as a nucleophile.

Loss of polymerase activity due to Tyr to Phe substitution in the YMDD motif of human immunodeficiency virus type-1 reverse transcriptase is compensated by Met to Val substitution within the same motif.

Tyr183 is a constituent of the highly conserved YXDD motif common to all retroviral reverse transcriptases. The two aspartates in this motif are the crucial members of the catalytic carboxylate triad while residue X, which in the case of HIV-1 RT is Met184, is implicated in dNTP substrate recognition and fidelity of DNA synthesis. In an attempt to understand the function of Tyr183 in the catalytic mechanism, we generated mutants of this residue (Y183F and Y183A) and subjected them to in-depth analysis. The efficiency of reverse transcription of natural U5-PBS HIV-1 RNA template was severely impaired by both the conservative and nonconservative substitutions. The major defect identified was at the level of dNTP binding as determined by a 20-80-fold increase in the Km for the dNTP substrate on both homopolymeric and heteropolymeric RNA and DNA templates. A significant reduction in processivity of DNA synthesis by these mutants was also noted. However, the fidelity of DNA synthesis by the Y183F and Y183A mutants was increased significantly compared to the wild-type enzyme. Interestingly, the reduction in the polymerase activity due to single substitution of Tyr to Phe in the YMDD motif is compensated by a second substitution of Met to Val in the same motif, herein referred to as the FVDD. The loss of dNTP binding as well as decreased processivity of DNA synthesis exhibited by the Y183F mutant was also compensated by mutation at the second site. Curiously, the double mutant did not exhibit any synergistic effect in regard to fidelity of DNA synthesis as might be expected since both the single mutations (Y183F, M184V) exhibited enhanced fidelity compared to the wild-type enzyme. These data implicate Tyr183 and Met184 as important constituents of the dNTP-binding pocket. We propose a model which suggests that subtle structural changes due to mutation in the flexible beta9-beta10 loop region at the active site of the molecule influence the enzyme activity and substrate recognition.

Active-site mobility in human immunodeficiency virus, type 1, protease as demonstrated by crystal structure of A28S mutant.

The mutation Ala28 to serine in human immunodeficiency virus, type 1, (HIV-1) protease introduces putative hydrogen bonds to each active-site carboxyl group. These hydrogen bonds are ubiquitous in pepsin-like eukaryotic aspartic proteases. In order to understand the significance of this difference between HIV-1 protease and homologous, eukaryotic aspartic proteases, we solved the three-dimensional structure of A28S mutant HIV-1 protease in complex with a peptidic inhibitor U-89360E. The structure has been determined to 2.0 A resolution with an R factor of 0.194. Comparison of the mutant enzyme structure with that of the wild-type HIV-1 protease bound to the same inhibitor (Hong L, Treharne A, Hartsuck JA, Foundling S, Tang J, 1996, Biochemistry 35:10627-10633) revealed double occupancy for the Ser28 hydroxyl group, which forms a hydrogen bond either to one of the oxygen atoms of the active-site carboxyl or to the carbonyl oxygen of Asp30. We also observed marked changes in orientation of the Asp25 catalytic carboxyl groups, presumably caused by the new hydrogen bonds. These observations suggest that catalytic aspartyl groups of HIV-1 protease have significant conformational flexibility unseen in eukaryotic aspartic proteases. This difference may provide an explanation for some unique catalytic properties of HIV-1 protease.

Electronic structure of axially ligated rhodium carboxylates: π back-bonding revisited

Fenske-Hall molecular orbital calculations have been applied to a new, structurally isolable analog of the intermediate involved in catalytic rhodium carboxylate carbenoid transformations. Results from the structural characterization of axially ligated rhodium acetate phenylisonitrile complexes have been utilized in approximate molecular orbital calculations. The results from the calculations suggest that a significant degree of π back-bonding exists between the metal and isonitrile fragments which, by analogy, implies that π back-bonding should also exist in the rhodium carbenoid intermediate. Sensitivity of the Fenske-Hall method to the magnitude of π back-donation in these complexes was gauged through calculations involving different phenylisonitrile groups in which the π back-bonding ability was modulated through derivativization. The reliability of the Fenske-Hall method was evaluated through a comparison to a high-level calculation.

Mutational analysis eliminates Glu 64 and Glu 94 as candidates for `catalytic carboxylate' in the bacterial ATP-binding-cassette protein MalK

MalK is the ATP-hydrolyzing subunit of the binding protein-dependent ATP-binding-cassette (ABC) transport system for maltose from Salmonella typhimurium. In a recent hypothesis, Glu 64 and Glu 94 of MalK were proposed as candidates for `catalytic carboxylate', common to ATP- and GTP-hydrolyzing proteins [Yoshida and Amano (1995) FEBS Lett. 359, 1–5]. Substitution of both residues and, additionally, Glu 74 by either glutamine or glycine and valine, respectively, had no deleterious effect on maltose transport. Thus, our data disprove the above notion.

Ionization states of the catalytic residues in HIV-1 protease.

Chemical synthesis was used to prepare the HIV-1 protease specifically 13C-labelled in the catalytically essential Asp 25 in each monomer. The NMR chemical shift of the 13C-enriched homodimeric enzyme was measured in the presence of the inhibitor pepstatin, a mimic of the tetrahedral intermediate formed in enzyme catalysis. In this complex, the catalytic carboxyls do not titrate in the pH range where the enzyme is active; throughout the range pH 2.5-6.5, one Asp 25 side chain is protonated and the other deprotonated. By contrast, in the absence of inhibitor the two Asp side chains are chemically equivalent and both deprotonated at pH6, the optimum for enzymatic activity. These direct observations of the chemical properties of the catalytic apparatus of the enzyme provide concrete information on which to base the design of improved HIV-1 protease inhibitors.

Characterization of a Potential Catalytic Residue, Asp-133, in the High Affinity ATP-binding Site of Escherichia coli SecA, Translocation ATPase

The high affinity ATP-binding site of SecA is located in its amino-terminal domain possessing amino acid sequences, the Walker A (GXXXXGKT) and B (ZZZZD) motifs, that are characteristic of a major class of nucleotide-binding sites (Walker, J. E., Saraste, M., Runswick, M. J., and Gay, N. J. (1982) EMBO J. 1, 945-951). Recently, we proposed that proteins possessing a typical set of Walker A and B motifs contain a conserved Glu or Asp between the two motifs. This Glu or Asp acts as a "catalytic residue" that activates a water molecule for an in-line attack on the gamma-phosphate of ATP (Amano, T., Yoshida, M., Matsuo, Y., and Nishikawa, K.(1995) FEBS Lett. 359, 1-5). In the present study, the aspartate residue at position 133 in Escherichia coli SecA, which could be the "catalytic residue," was mutated to an asparagine. The mutant SecA (SecA D133N) protein was expressed in E. coli CK4706, encoding a duplication of the secA gene, and purified to homogeneity. The in vitro protein translocation activity and membrane vesicle stimulated ATPase activity of SecA D133N were drastically reduced. Proteolytic studies indicated that the conformational changes of the mutant SecA occurring on interaction with ATP, presecretory proteins, phospholipids, and membrane vesicles, were similar to those of wild-type SecA. The mutant SecA allowed the signal peptide cleavage of proOmpA during translocation, indicating that the mutant retains the ability to bind ATP to perform the initial step of the translocation reaction. These data indicate that the carboxyl group of Asp-133 plays a role as a catalytic carboxylate, which activates a water molecule to attack gamma-phosphate of ATP, and the mutant lacking this residue cannot perform the total translocation but can still perform the initial step of the protein translocation.

Structure determination and refinement of the Humicola insolens endoglucanase V at 1.5 A resolution.

The structure of the catalytic core of the endoglucanase V (EGV) from Humicola insolens has been determined by the method of multiple isomorphous replacement at 1.5 A resolution. The final model, refined with X-PLOR and PROLSQ, has a crystallographic R factor of 0.163 (R(free) = 0.240) with deviations from stereochemical target values of 0.012 A and 0.037 degrees for bonds and angles, respectively. The model was further refined with SHELXL, including anisotropic modelling of the protein-atom temperature factors, to give a final model with an R factor of 0.105 and an R(free) of 0.154. The initial isomorphous replacement electron-density map was poor and uninterpretable but was improved by the use of synchrotron data collected at a wavelength chosen so as to optimize the f" contribution of the anomalous scattering from the heavy atoms. The structure of H. insolens EGV consists of a six-stranded beta-barrel domain, similar to that found in a family of plant defence proteins, linked by a number of disulfide-bonded loop regions. A long open groove runs across the surface of the enzyme either side of which lie the catalytic aspartate residues. The 9 A separation of the catalytic carboxylate groups is consistent with the observation that EGV catalyzes the hydrolysis of the cellulose, beta(1-->4) links with inversion of configuration at the anomeric C1 atom. This structure is the first representative from the glycosyl hydrolase family 45.

Functional role of the active site glutamate-368 in rat short chain acyl-CoA dehydrogenase.

The acyl-CoA dehydrogenases are a family of flavoenzymes with similar structure and function involved in the metabolism of fatty acids and branched chain amino acids. The degree of overlap in substrate specificity is narrow among these enzymes. The position of the catalytic glutamate, identified as Glu376 in porcine medium chain acyl-CoA dehydrogenase (MCAD), Glu254 in human isovaleryl-CoA dehydrogenase (IVD), and Glu261 in human long chain acyl-CoA dehydrogenase (LCAD), has been suggested to affect substrate chain length specificity. In this study, in vitro site-directed mutagenesis was used to investigate the effect of changing the position of the catalytic carboxylate on substrate specificity in short chain acyl-CoA dehydrogenase (SCAD). Glu368, the hypothetical active site catalytic residue of rat SCAD, was replaced with Asp, Gly, Gln, Arg, and Lys and the wild type and mutant SCADs were produced in Escherichia coli and purified. The recombinant wild type SCAD kcat/K(m) values for butyryl-hexanoyl-, and octanoyl-CoA were 220, 22, and 3.2 microM-1 min-1, respectively, while the Glu368Asp mutant gave kcat/K(m) of 81, 12, and 1.4 microM-1 min-1, respectively, for the same substrates. None of the other mutants exhibited enzyme activity. A Glu368Gly/Gly247Glu double mutant enzyme, which places the catalytic residue at a position homologous to that of LCAD, was also synthesized and purified. It showed kcat/K(m) of 9.3, 2.8, and 1.5 microM-1 min-1 with butyryl-, hexanoyl-, and octanoyl-CoA used as substrates, respectively. These results confirm the identity of Glu368 as the catalytic residue of rat SCAD and suggest that alteration of the position of the catalytic carboxylate can modify substrate specificity.

Structure of binary and ternary complexes of zinc and cobalt carboxypeptidase A as determined by X-ray absorption fine structure.

Carboxypeptidase A, ZnCPD, is typical of a wide range of exo- and endo-metalloproteases that have three protein ligands and a water molecule bound to a catalytic zinc atom and a glutamate residue in the active site that likely acts in conjunction with the Zn-bound water to bring about catalysis. Such enzymes generally have bell-shaped pH-activity profiles (EH2 in equilibrium with EH in equilibrium with E) where the concentration of the catalytic species EH is regulated by pKEH2 and pKEH. The present X-ray absorption fine structure, XAFS, study has determined the structure and pH behavior of the binary and ternary product complexes in order to examine the role of the Zn-bound water in catalysis. Increasing the pH from 7 to 10 of the ZnCPD.L-Phe complex results in the same type of progressive spectral changes in the near-edge XAFS spectrum as is seen for ZnCPD, but the changes are complete by more than 1 pH value below that observed for ZnCPD. The results are in agreement with kinetic studies that show E binds the protonated form of L-Phe more tightly than EH, thus in effect decreasing the value of pKEH. The XAFS results show the average interatomic distance. R. for the zinc ligands of the EH.L-Phe complex decreases by 0.02 A upon formation of the E.L-Phe complex, essentially identical to that obtained for the EH and E forms of the native enzyme. Addition of azide to ZnCPD.L-Phe at pH 7 markedly changes the zinc coordination sphere from 4 N/O atoms at 2.021 +/- O.06 A and 1.4 +/- 0.5 N/O atoms at 2.54 +/- 0.5 A to 3.9 N/O atoms at 1.995 +/- 0.006 A. The decrease in R of about 0.03 A in both the Zn- and CoCPD.L-Phe.N3-complexes is likely due to the ligand exchange from a neutral water to an anion. The XAFS spectra of the ternary complex is pH independent from 7 to 9, in agreement with the ionization of the water being the source of the spectral changes in the free enzyme and its binary L-Phe complex. The enzyme.azide.L-Phe complex is likely bound in a manner analogous to that expected for a post-transition state in a biproduct complex for peptide hydrolysis--that is, the carboxylate anion of the peptide bound to the Zn and the protonated form of L-Phe H-bonded to the catalytic Glu-270 carboxylate. The XAFS results on the "spectroscopically silent" ZnCPD are compared to nuclear magnetic resonance and electron absorption studies on CoCPD.

HO. and DNase I probing of E sigma 70 RNA polymerase--lambda PR promoter open complexes: Mg2+ binding and its structural consequences at the transcription start site.

Chemical and enzymatic probing (footprinting) of the reactivity of the promoter DNA backbone is applied to characterize two binary open complexes RPo1 (-Mg2+) and RPo2 (+Mg2+), formed by Escherichia coli RNA polymerase (E sigma 70) at the lambda PR promoter. We report that HO. detects major differences in backbone reactivity between RPo1 and RPo2 in the open region from -4 to +1 relative to the transcription start site. Deoxyribose sugars at positions -4 to +1 of the t (template) strand react with HO. in RPo2 but are relatively protected in RPo1. Binding of Mg2+ to convert RPo1 to RPo2 therefore increases the reactivity of two negatively charged footprinting agents [MnO4-; Suh, W.-C., Ross, W., & Record, M. T., Jr., (1993) Science 259, 358-361; and Fe(EDTA)2-/HO.] at the start site and is required for binding of the negatively-charged initiating nucleotides to the polymerase and the t strand at the start site. We propose that these effects result from binding of two Mg2+ ions to the catalytic carboxyls in the nucleotide binding sites. Except for the key region on the t strand at the start site, the promoter DNA of both RPo1 and RPo2 is continuously protected from DNase I and hydroxyl radical (HO.) cleavage between the -12 and +25 promoter positions. Protection in the upstream region, extending from -13 to about -70, is periodic, with an 11 base pair periodicity indicative of binding of polymerase to a single face of the DNA helix.(ABSTRACT TRUNCATED AT 250 WORDS)

High-resolution Structure of the Catalytic Domain of Avian Sarcoma Virus Integrase

Retroviral integrase (IN) functions to insert retroviral DNA into the host cell chromosome in a highly coordinated manner. IN catalyzes two biochemically separable reactions: processing of the viral DNA ends and joining of these ends to the host DNA. Previous studies suggested that these two reactions are chemically similar and are carried out by a single active site that is characterized by a highly conserved constellation of carboxylate residues, the D,D(35)E motif. We report here the crystal structure of the isolated catalytic domain of avian sarcoma virus (ASV) IN, solved using multiwavelength anomalous diffraction data for a selenomethionine derivative and refined at 1.7 Å resolution. The protein is a crystallographic dimer with each monomer featuring a five-stranded mixed β-sheet region surrounded by five α-helices. Based on the general fold and the arrangement of catalytic carboxylate residues, it is apparent that ASV IN is a member of a superfamily of proteins that also includes two types of nucleases, RuvC and RNase H. The general fold and the dimer interface are similar to those of the analogous domain of HIV-1 IN, whose crystal structure has been determined at 2.5 Å resolution. However, the ASV IN structure is more complete in that all three critical carboxylic acids, Asp64, Asp121 and Glu157, are ordered. The ordered active site and the considerably higher resolution of the present structure are all important to an understanding of the mechanism of retroviral DNA integration, as well as for designing antiviral agents that may be effective against HIV.

Spatial precision of a catalytic carboxylate of F 1-ATPase β subunit probed by introducing different carboxylate-containing side chains

Combining mutation and chemical modification, we have introduced Asp, Gln, Cys, S-carboxymethylcysteine (Cax) and S-carbamoylmethylcysteine (Cam) into the positions of Glu 190 and Glu 201 of the β subunit of F 1-ATPase from the thermophilic Bacillus PS3. The steady-state ATPase activities of α 3β 3γ complexes containing these changed β subunits were 12% (E190Cax), 7% (E190D), 3% (E190Cam), <1% (E190C), <1% (E190Q), and 73% (E201D), 40% (E201Cax), 25% (E201C), 20% (E201Q), 4% (E201Cam), of that of the wild-type α 3β 3γ complex. For the complexes containing E190C or E190Q, even the ability of single-site catalysis was lost. Thus, the presence of a carboxylate at 190 (but not at 201) is absolutely required for catalysis and its spatial precision is very strict. Analysis of inactivation of the complexes by dicyclohexylcarbodiimide suggests that Glu 190 and Glu 201 are interacting in the F 1-ATPase.

X-ray Analysis at 2·0 Å Resolution of Mouse Submaxillary Renin Complexed with a Decapeptide Inhibitor CH-66, Based on the 4-16 Fragment of Rat Angiotensinogen

The structure of mouse submaxillary renin complexed with a decapeptide inhibitor, CH-66 (Piv-His-Pro-Phe-His-Leu-OH-Leu-Tyr-Tyr-Ser-NH 2), where Piv denotes a pivaloyl blocking group, and -OH- denotes a hydroxyethylene (-(S)CHOH-CH 2-) transition state isostere as a scissile bond surrogate, has been refined to an agreement factor of 0·18 at 2·0 Å resolution. The positions of 10,038 protein atoms and 364 inhibitor atoms (4 independent protein inhibitor complexes), as well as of 613 solvent atoms, have been determined with an estimated root-mean-square (r.m.s.) error of 0·21 Å. The r.m.s. deviation from ideality for bond distances is 0·026 Å, and for angle distances is 0·0543 Å. We have compared the three-dimensional structure of mouse renin with other aspartic proteinases, using rigid-body analysis with respect to shifts involving the domain comprising residues 190 to 302. In terms of the relative orientation of domains, mouse submaxillary renin is closest to human renin with only a 1·7° difference in domain orientation. Porcine pepsin (the molecular replacement model) differs structurally from mouse renin by a 6·9° domain rotation, whereas endothiapepsin, a fungal aspartic proteinase, differs by 18·8°. The triple proline loop (residues 292 to 294), which is structurally opposite the active-site "flap" (residues 72 to 83), gives renin a superficial resemblance to the fold of the retroviral proteinases. The inhibitor is bound in an extended conformation along the active-site cleft, and the hydroxyethylene moiety forms hydrogen bonds with both catalytic aspartate carboxylates. The complex is stabilized by hydrogen bonds between the main chain of the inhibitor and the enzyme. All side-chains of the inhibitor are in van der Waals contact with groups in the enzyme and define ten specificity sub-sites. This study shows how renin compact sub-sites due to the positioning of secondary structure elements, to complementary substitutions and to the residue composition of its loops close to the active site, leading to extreme specificity towards its prohormone substrate, angiotensinogen. We have analyzed the micro-environment of each of the buried charged groups in order to predict their ionization states.

Hydrolysis of glycosylpyridinium ions by anomeric-configuration-inverting glycosidases

The hydrolyses of five β- d-xylopyranosylpyridinium ions by the β- d-xylosidase of Bacillus pumilus proceed with k cat values 10 8–10 9-fold larger than the rates of spontaneous hydrolysis of the same compounds. Log( k cat) values correlate well with aglycon p K a [β 1g( V) = −0.52, r = 0.99], whereas the correlation of log( k cat/ K m) is poor [r = 0.77; β 1g( V/K) = ∼ −0.6]. The (1 → 3)-β- d-glucanase of Sporotrichum dimorphosporum hydrolyses 4-bromo-2-(β- d-glucopyranosyl)isoquinolinium ion with a rate enhancement of 10 8. The amyloglucosidase II of Aspergillus niger hydrolyses three α- d-gluco-pyranosylpyridinium ions with rate enhancements of 10 5–10 8. The efficient hydrolysis of glycosylpyridinium ions by these three inverting glycosidases, the catalytic mechanism of which is unlikely to involve a nucleophile from the enzyme, makes it imporable that the hydrolysis of glycosylpyridinium ions by retaining glycosidases discovered some years ago, is initiated by addition of a catalytic nucleophilic carboxylate group of the enzyme to the pyridinium ring.

Identification of glutamic acid 105 at the active site of Bacillus amyloliquefaciens 1,3-1,4-beta-D-glucan 4-glucanohydrolase using epoxide-based inhibitors.

Bacillus amyloliquefaciens 1,3-1,4-beta-D-glucan 4-glucanohydrolase (EC 3.2.1.73) was modified by the mechanism-based, affinity-labeling reagent [14C](3,4)-epoxybutyl beta-D-cellobioside. Following partial inactivation a completely inactivated enzyme preparation containing 1.1 mol of covalently bound inhibitor/mol of protein was obtained by chromatography on a cellulosic matrix. The inactivated enzyme was digested with endoproteinase Glu-C and radioactive peptides purified by reversed-phase high performance liquid chromatography (HPLC). The affinity label was esterified exclusively to the gamma-carboxylate of Glu105 in the sequence Gly-Thr-Pro-Trp-Asp-Glu-Ile-Asp-Ile-Glu109. The sequence motif Glu-(Ile/Leu)-Asp-Ile is found in many glucanases and xylanases and may therefore serve to identify the catalytic nucleophile in beta-glycanases, which otherwise exhibit a low degree of sequence identity. The esterification of Glu105 by the affinity label abolished endoproteinase Glu-C-mediated hydrolysis of the Glu-Ile106 peptide bond. Identification of phenylthiohydantoin-Glu105 during automated sequence analysis was not possible unless the affinity label was liberated by prior base hydrolysis. These observations formed the basis for the development of a highly sensitive approach for the identification of catalytic carboxylates in polysaccharide hydrolases employing non-radioactive inhibitors, comparative HPLC mapping, electrospray mass spectrometry, and Edman degradation.

Multiple role of hydrophobicity of tryptophan-108 in chicken lysozyme: structural stability, saccharide binding ability, and abnormal pKa of glutamic acid-35.

Trp108 of chicken lysozyme is in van der Waals contact with Glu35, one of two catalytic carboxyl groups. The role of Trp108 in lysozyme function and stability was investigated by using mutant lysozymes secreted from yeast. By the replacement of Trp108 with less hydrophobic residues, Tyr (W108Y lysozyme) and Gln (W108Q lysozyme), the activity, saccharide binding ability, stability, and pKa of Glu35 were all decreased with a decrease in the hydrophobicity of residue 108. Namely, at pH 5.5 and 40 degrees C, the activities of W108Y and W108Q lysozymes against glycol chitin were 17.3 and 1.6% of that of wild-type lysozyme, and their dissociation constants for the binding of a trimer of N-acetyl-D-glucosamine were 7.4 and 309 times larger than that of wild-type lysozyme, respectively. For the reversible unfolding at pH 3.5 and 30 degrees C, W108Y and W108Q lysozymes were less stable than wild-type lysozyme by 1.4 and 3.6 kcal/mol, respectively. As for the pKa of Glu35, the values for W108Y and W108Q lysozymes were found to be lower than that for wild-type lysozyme by 0.2 and by 0.6 pKa unit, respectively. The pKa of Glu35 in lysozyme was also decreased from 6.1 to 5.4 by the presence of 1-3 M guanidine hydrochloride, or to 5.5 by the substitution of Asn for Asp52, another catalytic carboxyl group. Thus, both the hydrophobicity of Trp108 and the electrostatic interaction with Asp52 are equally responsible for the abnormally high pKa (6.1) of Glu35, compared with that (4.4) of a normal glutamic acid residue.(ABSTRACT TRUNCATED AT 250 WORDS)

Pancreatic lipases: evolutionary intermediates in a positional change of catalytic carboxylates?

Comparison of the fold of lipases from Geotrichum candidum and from human pancreas identified a high degree of similarity which was not expected on the basis of their amino acid sequences. Although both enzymes utilize a serine protease-like catalytic triad, they differ in the topological position of the acid. We speculate that these proteins are evolutionarily related and that the pancreatic lipase is an evolutionary intermediate in the pathway of migration of the catalytic acid to a new position within the fold.