Biochemical characteristics of C-terminal region of recombinant chitinase from Bacillus licheniformis: implication of necessity for enzyme properties

The functional and structural significance of the C-terminal region of Bacillus licheniformis chitinase was explored using C-terminal truncation mutagenesis. Comparative studies between full-length and truncated mutant molecules included initial rate kinetics, fluorescence and CD spectrometric properties, substrate binding and hydrolysis abilities, thermostability, and thermodenaturation kinetics. Kinetic analyses revealed that the overall catalytic efficiency, k(cat)/K(m), was slightly increased for the truncated enzymes toward the soluble 4-methylumbelliferyl-N-N'-diacetyl chitobiose or 4-methylumbelliferyl-N-N'-N'-triacetyl chitotriose or insoluble alpha-chitin substrate. By contrast, changes to substrate affinity, K(m), and turnover rate, k(cat), varied considerably for both types of chitin substrates between the full-length and truncated enzymes. Both truncated enzymes exhibited significantly higher thermostabilities than the full-length enzyme. The truncated mutants retained similar substrate-binding specificities and abilities against the insoluble substrate but only had approximately 75% of the hydrolyzing efficiency of the full-length chitinase molecule. Fluorescence spectroscopy indicated that both C-terminal deletion mutants retained an active folding conformation similar to the full-length enzyme. However, a CD melting unfolding study was able to distinguish between the full-length and truncated mutant molecules by the two phases of apparent transition temperatures in the mutants. These results indicate that up to 145 amino acid residues, including the putative C-terminal chitin-binding region and the fibronectin (III) motif of B. licheniformis chitinase, could be removed without causing a seriously aberrant change in structure and a dramatic decrease in insoluble chitin hydrolysis. The results of the present study provide evidence demonstrating that the binding and hydrolyzing of insoluble chitin substrate for B. licheniformis chitinase was not dependent solely on the putative C-terminal chitin-binding region and the fibronectin (III) motif.     

Mutations in the charged residues of the amino terminus of rat liver fructose 6-phosphate,2-kinase:Fructose 2,6-bisphosphatase: effects on regulation.

Amino and carboxyl termini of the bifunctional enzyme Fru 6-P, 2-kinase:Fru 2,6-bisphosphatase regulate the relative activities of the kinase/phosphatase. The N-terminus of the rat liver bifunctional enzyme is highly basic, containing a protein kinase A phosphorylation site that regulates these enzyme activities in a reciprocal manner. To determine the role of charged residues in the N-terminal peptide, mutant enzymes were constructed in which these residues were altered to residues carrying opposite charges, and the effect on the catalytic properties, thermal lability, and susceptibility to trypsin digestion and phosphorylation by protein kinase A was determined. Most of these mutations decreased k(cat)/K(ATP) and/or k(cat)/K(Fru) (6-P) of the kinase and increased k(cat)/K(Fru 2,6-P2) of the phosphatase. These mutant enzymes were more susceptible to trypsin digestion, phosphorylation by protein kinase A, and thermal inactivation. In general, the effect was greater with amino acid residues located more distant from the N-terminus. The resulting changes were not as large as observed with the phosphorylated enzyme. Mutation of Ser22 to Pro produced large changes in the kinetic properties comparable to those of phosphorylation, suggesting that the flexible region of the N-terminus containing five serines (Ser20 to S24) is essential for the enzyme activities. These results indicated that the charged residues as well as Ser20-Ser24 in the N-terminus of the liver Fru 6-P,2-kinase:Fru 2,6-Pase are essential in the allosteric regulation and probably involved in interactions with the catalytic domains that induce a conformation that has high Fru 6-P,2-kinase and low Fru 2,6-Pase activities. Any disruption of this N-terminal interaction results in inhibition of the kinase and activation of the phosphatase.     

Probing the role of the C-terminus of Bacillus subtilis chorismate mutase by a novel random protein-termination strategy

A novel strategy combining random protein truncation and genetic selection has been developed to identify dispensable C-terminal segments of an enzyme. This approach, which entails the random introduction of premature termination codons, was applied to the last 17 residues of chorismate mutase from Bacillus subtilis (BsCM). Although structurally ill-defined, the C-terminus of BsCM has been proposed to cap the active site upon substrate binding and affect catalysis. However, sequence patterns of 178 selected gene variants show that the final 11 residues of the protein can be mutated and even removed without significantly impairing activity in vivo. In fact, none of the randomized residues is absolutely required, but a preference for wild-type Lys111, Ala112, Leu115, and Arg116 is apparent. These residues are part of a C-terminal 3(10)-helix and provide contacts with the rest of the protein or its ligands. The kinetic parameters of selected enzyme variants show that truncations and mutations do not significantly impair catalytic turnover (k(cat)) but substantially decrease k(cat)/K(m). Thus, while the 17 C-terminal residues of BsCM do not participate directly in the chemical rearrangement, they appear to contribute to enzymatic efficiency via uniform binding of the substrate and transition state.     

The carboxyl terminus of coffee bean alpha-galactosidase is critical for enzyme activity

The role of the carboxyl (C)-terminal region of coffee bean alpha-galactosidase (alpha-GAL) has been studied by expressing C-terminal deletion mutants in the methylotrophic yeast strain Pichia pastoris. A previous study of human alpha-galactosidase determined that enzyme activity increased when up to 10 amino acid residues were deleted. Deleting 11 residues reduced activity, and deleting 12 residues abolished activity. In our studies, alpha-GAL activity is reduced when one or two amino acids are deleted, as is enzyme secretion directed by P. pastoris signal sequences. The pH profile is similar to that of the wild-type enzyme. Deleting 3 or more residues from the C-terminal end results in a complete loss of both enzyme secretion and activity. The C-terminus of alpha-GAL seems to play an important role in overall enzyme conformation and may directly affect the proper conformation of the active site.     

Improving the activity and stability of GL-7-ACA acylase CA130 by site-directed mutagenesis

In the present study, glutaryl-7-amino cephalosporanic acid acylase from Pseudomonas sp. strain 130 (CA130) was mutated to improve its enzymatic activity and stability. Based on the crystal structure of CA130, two series of amino acid residues, one from those directly involved in catalytic function and another from those putatively involved in surface charge, were selected as targets for site-directed mutagenesis. In the first series of experiments, several key residues in the substrate-binding pocket were substituted, and the genes were expressed in Escherichia coli for activity screening. Two of the mutants constructed, Y151alphaF and Q50betaN, showed two- to threefold-increased catalytic efficiency (k(cat)/K(m)) compared to wild-type CA130. Their K(m) values were decreased by ca. 50%, and the k(cat) values increased to 14.4 and 16.9 s(-1), respectively. The ability of these mutants to hydrolyze adipoyl 6-amino penicillinic acid was also improved. In the second series of mutagenesis, several mutants with enhanced stabilities were identified. Among them, R121betaA and K198betaA had a 30 to 58% longer half-life than wild-type CA130, and K198betaA and D286betaA showed an alkaline shift of optimal pH by about 1.0 to 2.0 pH units. To construct an engineered enzyme with the properties of both increased activity and stability, the double mutant Q50betaN/K198betaA was expressed. This enzyme was purified and immobilized for catalytic analysis. The immobilized mutant enzyme showed a 34.2% increase in specific activity compared to the immobilized wild-type CA130.     

The bifunctional active site of S-adenosylmethionine synthetase. Roles of the basic residues

S-adenosylmethionine (AdoMet) synthetase catalyzes a unique two-step enzymatic reaction leading to formation of the primary biological alkylating agent. The crystal structure of Escherichia coli AdoMet synthetase shows that the active site, which lies between two subunits, contains four lysines and one histidine as basic residues. In order to test the proposed charge and hydrogen bonding roles in catalytic function, each lysine has been changed to an uncharged methionine or alanine, and the histidine has been altered to asparagine. The resultant enzyme variants are all tetramers like the wild type enzyme; however, circular dichroism spectra show reductions in helix content for the K245*M and K269M mutants. (The asterisk denotes that the residue is in the second subunit.) Four mutants have k(cat) reductions of approximately 10(3)-10(4)-fold in AdoMet synthesis; however, the k(cat) of K165*M variant is only reduced 2-fold. In each mutant, there is a smaller catalytic impairment in the partial reaction of tripolyphosphate hydrolysis. The K165*A enzyme has a 100-fold greater k(cat) for tripolyphosphate hydrolysis than the wild type enzyme, but this mutant is not activated by AdoMet in contrast to the wild type enzyme. The properties of these mutants require reassessment of the catalytic roles of these residues.     

The N-terminal domain of the regulatory subunit is sufficient for complete activation of acetohydroxyacid synthase III from Escherichia coli

We have previously proposed a model for the fold of the N-terminal domain of the small, regulatory subunit (SSU) of acetohydroxyacid synthase isozyme III. The fold is an alpha-beta sandwich with betaalphabetabetaalphabeta topology, structurally homologous to the C-terminal regulatory domain of 3-phosphoglycerate dehydrogenase. We suggested that the N-terminal domains of a pair of SSUs interact in the holoenzyme to form two binding sites for the feedback inhibitor valine in the interface between them. The model was supported by mutational analysis and other evidence. We have now examined the role of the C-terminal portion of the SSU by construction of truncated polypeptides (lacking 35, 48, 80, 95, or 112 amino acid residues from the C terminus) and examining the properties of holoenzymes reconstituted using these constructs. The Delta35, Delta48, and Delta80 constructs all lead to essentially complete activation of the catalytic subunits. The Delta80 construct, corresponding to the putative N-terminal domain, has the highest level of affinity for the catalytic subunits and leads to a reconstituted enzyme with k(cat)/K(M) about twice that of the wild-type enzyme. On the other hand, none of these constructs binds valine or leads to a valine-sensitive enzyme on reconstitution. The enzyme reconstituted with the Delta80 construct does not bind valine, either. The N-terminal portion (about 80 amino acid residues) of the SSU is thus necessary and sufficient for recognition and activation of the catalytic subunits, but the C-terminal half of the SSU is required for valine binding and response. We suggest that the C-terminal region of the SSU contributes to monomer-monomer interactions, and provide additional experimental evidence for this suggestion.     

Structure and catalytic cycle of beta-1,4-galactosyltransferase

Beta-1,4-galactosyltransferase-1, a housekeeping enzyme that functions in the synthesis of glycoconjugates, has two flexible loops, one short and one long. Upon binding a metal ion and UDP-galactose, the loops change from an open to a closed conformation, repositioning residues to lock the ligands in place. Residues at the N-terminal region of the long loop form the metal-binding site and those at the C-terminal region form a helix, which becomes part of the binding site for the oligosaccharide acceptor; the remaining residues cover the bound sugar-nucleotide. After binding of the oligosaccharide acceptor and transfer of the galactose moiety, the product disaccharide unit is ejected and the enzyme returns to the open conformation, repeating the catalytic cycle.     

Recombinant collagen studies link the severe conformational changes induced by osteogenesis imperfecta mutations to the disruption of a set of interchain salt bridges

The clinical severity of Osteogenesis Imperfecta (OI), also known as the brittle bone disease, relates to the extent of conformational changes in the collagen triple helix induced by Gly substitution mutations. The lingering question is why Gly substitutions at different locations of collagen cause different disruptions of the triple helix. Here, we describe markedly different conformational changes of the triple helix induced by two Gly substitution mutations placed only 12 residues apart. The effects of the Gly substitutions were characterized using a recombinant collagen fragment modeling the 63-residue segment of the alpha1 chain of type I collagen containing no Hyp (residues 877-939) obtained from Escherichia coli. Two Gly --> Ser substitutions at Gly-901 and Gly-913 associated with, respectively, mild and severe OI variants were introduced by site-directed mutagenesis. Biophysical characterization and limited protease digestion experiments revealed that while the substitution at Gly-901 causes relatively minor destabilization of the triple helix, the substitution at Gly-913 induces large scale unfolding of an unstable region C-terminal to the mutation site. This extensive unfolding is caused by the intrinsic low stability of the C-terminal region of the helix and the mutation induced disruption of a set of salt bridges, which functions to lock this unstable region into the triple helical conformation. The extensive conformational changes associated with the loss of the salt bridges highlight the long range impact of the local interactions of triple helix and suggest a new mechanism by which OI mutations cause severe conformational damages in collagen.     

Cytoplasmic aspartyl-tRNA synthetase from Saccharomyces cerevisiae. Study of its functional organisation by deletion analysis

Aspartyl-tRNA synthetase (AspRS) from yeast, a homodimer of 125 kDa, was shortened by several residues from the C- and N-termini, via site-directed mutagenesis, to examine the contribution of the removed peptides to the enzyme properties. This study showed that the N-terminal sequence up to amino acid 70 (which confers peculiar ionic properties to the protein) is dispensable for activity. Domains located beyond amino acid 70 appeared to have increasing catalytic importance; the removal of 80 or 90 residues affected the Km values for ATP and deletions of 101 or 140 amino acids profoundly modified the physiochemical properties of AspRS, and by consequence, its structural organisation (extraction of the mutated proteins out of the cells required the presence of SDS). On the C-terminal side, very limited modifications readily affected the enzyme properties. Deletion of as few as three residues increased the Km for ATP and reduced the aminoacylation kcat as well as the thermostability of the adenylate synthesis activity; the kcat of this step was impaired after deletion of two further residues. Finally, shortening the C-terminal decapeptide completely inactivated AspRS, whilst affecting neither its affinity for tRNAAsp nor its dimerisation capacity. These data reveal the role of the C-terminal decapeptide as a determinant in both reactions catalysed by AspRS. This peptide is involved in ATP binding, stabilising the functional conformation of the amino-acid-activating domain and probably maintaining the tRNA-acceptor end in a reactive position with regard to the activated amino acid.     

A structure-function study of a proton transport pathway in the gamma-class carbonic anhydrase from Methanosarcina thermophila

Four glutamate residues in the prototypic gamma-class carbonic anhydrase from Methanosarcina thermophila (Cam) were characterized by site-directed mutagenesis and chemical rescue studies. Alanine substitution indicated that an external loop residue, Glu 84, and an internal active site residue, Glu 62, are both important for CO(2) hydration activity. Two other external loop residues, Glu 88 and Glu 89, are less important for enzyme function. The two E84D and -H variants exhibited significant activity relative to wild-type activity in pH 7.5 MOPS buffer, suggesting that the original glutamate residue could be substituted with other ionizable residues with similar pK(a) values. The E84A, -C, -K, -Q, -S, and -Y variants exhibited large decreases in k(cat) values in pH 7.5 MOPS buffer, but only exhibited small changes in k(cat)/K(m). These same six variants were all chemically rescued by pH 7.5 imidazole buffer, with 23-46-fold increases in the apparent k(cat). These results are consistent with Glu 84 functioning as a proton shuttle residue. The E62D variant exhibited a 3-fold decrease in k(cat) and a 2-fold decrease in k(cat)/K(m) relative to those of the wild type in pH 7.5 MOPS buffer, while other substitutions (E62A, -C, -H, -Q, -T, and -Y) resulted in much larger decreases in both k(cat) and k(cat)/K(m). Imidazole did not significantly increase the k(cat) values and slightly decreased the k(cat)/K(m) values of most of the Glu 62 variants. These results indicate a primary preference for a carboxylate group at position 62, and support a proposed catalytic role for residue Glu 62 in the CO(2) hydration step, but do not definitively establish its role in the proton transport step.     

Roles of substrate distortion and intramolecular hydrogen bonding in enzymatic catalysis by scytalone dehydratase.

Alternative substrates and site-directed mutations of active-site residues are used to probe factors controlling the catalytic efficacy of scytalone dehydratase. In the E1cb-like, syn-elimination reactions catalyzed, efficient catalysis requires distortion of the substrate ring system to facilitate proton abstraction from its C2 methylene and elimination of its C3 hydroxyl group. Theoretical calculations indicate that such distortions are more readily achieved in the substrate 2,3-dihydro-2,5-dihydroxy-4H-benzopyran-4-one (DDBO) than in the physiological substrates vermelone and scytalone by approximately 2 kcal/mol. A survey of 12 active-site amino acid residues reveals 4 site-directed mutants (H110N, N131A, F53A, and F53L) have higher relative values of k(cat) and k(cat)/K(m) for DDBO over scytalone and for DDBO over vermelone than the wild-type enzyme, thus suggesting substrate-distortion roles for the native residues in catalysis. A structural link for this function is in the modeled enzyme-substrate complex where F53 and H110 are positioned above and below the substrate's C3 hydroxyl group, respectively, for pushing and pulling the leaving group into the axial orientation of a pseudo-boat conformation; N131 hydrogen-bonds to the C8 hydroxyl group at the opposite end of the substrate, serving as a pivot for the actions of F53 and H110. Deshydroxyvermelone lacks the phenolic hydroxyl group and the intramolecular hydrogen bond of vermelone. The relative values of k(cat) (95) and k(cat)/K(m) (1800) for vermelone over deshydroxyvermelone for the wild-type enzyme indicate the importance of the hydroxyl group for substrate recognition and catalysis. Off the enzyme, the much slower rates for the solvolytic dehydration of deshydroxyvermelone and vermelone are similar, thus specifying the importance of the hydroxyl group of vermelone for enzyme catalysis.     

Swapping FAD binding motifs between plastidic and bacterial ferredoxin-NADP(H) reductases

Plant-type ferredoxin-NADP(H) reductases (FNRs) are grouped in two classes, plastidic with an extended FAD conformation and high catalytic rates and bacterial with a folded flavin nucleotide and low turnover rates. The 112-123 β-hairpin from a plastidic FNR and the carboxy-terminal tryptophan of a bacterial FNR, suggested to be responsible for the FAD differential conformation, were mutually exchanged. The plastidic FNR lacking the β-hairpin was unable to fold properly. An extra tryptophan at the carboxy terminus, emulating the bacterial FNR, resulted in an enzyme with decreased affinity for FAD and reduced diaphorase and ferredoxin-dependent cytochrome c reductase activities. The insertion of the β-hairpin into the corresponding position of the bacterial FNR increased FAD affinity but did not affect its catalytic properties. The same insertion with simultaneous deletion of the carboxy-terminal tryptophan produced a bacterial chimera emulating the plastidic architecture with an increased k(cat) and an increased catalytic efficiency for the diaphorase activity and a decrease in the enzyme's ability to react with its substrates ferredoxin and flavodoxin. Crystallographic structures of the chimeras showed no significant changes in their overall structure, although alterations in the FAD conformations were observed. Plastidic and bacterial FNRs thus reveal differential effects of key structural elements. While the 112-123 β-hairpin modulates the catalytic efficiency of plastidic FNR, it seems not to affect the bacterial FNR behavior, which instead can be improved by the loss of the C-terminal tryptophan. This report highlights the role of the FAD moiety conformation and the structural determinants involved in stabilizing it, ultimately modulating the functional output of FNRs.     

Site-directed mutagenesis of selected residues at the active site of aryl-alcohol oxidase, an H2O2-producing ligninolytic enzyme

Aryl-alcohol oxidase provides H(2)O(2) for lignin biodegradation, a key process for carbon recycling in land ecosystems that is also of great biotechnological interest. However, little is known of the structural determinants of the catalytic activity of this fungal flavoenzyme, which oxidizes a variety of polyunsaturated alcohols. Different alcohol substrates were docked on the aryl-alcohol oxidase molecular structure, and six amino acid residues surrounding the putative substrate-binding site were chosen for site-directed mutagenesis modification. Several Pleurotus eryngii aryl-alcohol oxidase variants were purified to homogeneity after heterologous expression in Emericella nidulans, and characterized in terms of their steady-state kinetic properties. Two histidine residues (His502 and His546) are strictly required for aryl-alcohol oxidase catalysis, as shown by the lack of activity of different variants. This fact, together with their location near the isoalloxazine ring of FAD, suggested a contribution to catalysis by alcohol activation, enabling its oxidation by flavin-adenine dinucleotide (FAD). The presence of two aromatic residues (at positions 92 and 501) is also required, as shown by the conserved activity of the Y92F and F501Y enzyme variants and the strongly impaired activity of Y92A and F501A. By contrast, a third aromatic residue (Tyr78) does not seem to be involved in catalysis. The kinetic and spectral properties of the Phe501 variants suggested that this residue could affect the FAD environment, modulating the catalytic rate of the enzyme. Finally, L315 affects the enzyme k(cat), although it is not located in the near vicinity of the cofactor. The present study provides the first evidence for the role of aryl-alcohol oxidase active site residues.     

Interconverting the catalytic activities of (betaalpha)(8)-barrel enzymes from different metabolic pathways: sequence requirements and molecular analysis

The (betaalpha)(8)-barrel enzymes N'-[(5'-phosphoribosyl)formimino]-5-aminoimidazole-4-carboxamide ribonucleotide isomerase (tHisA) and imidazole glycerol phosphate synthase (tHisF) from Thermotoga maritima catalyze two successive reactions in the biosynthesis of histidine. In both enzymes, aspartate residues at the C-terminal end of beta-strand 1 (Asp8 in tHisA and Asp11 in tHisF) and beta-strand 5 (Asp127 in tHisA and Asp130 in tHisF) are essential for catalytic activity. It was demonstrated earlier that in tHisA the substitution of Asp127 by valine (tHisA-D127V) generates phosphoribosylanthranilate isomerase (TrpF) activity, a related (betaalpha)(8)-barrel enzyme participating in tryptophan biosynthesis. It is shown here that in tHisF the corresponding substitution of Asp130 by valine (tHisF-D130V) also generates TrpF activity. To determine the effectiveness of individual amino acid exchanges in these conversions, each of the 20 standard amino acid residues was introduced at position 127 of tHisA and 130 of tHisF by saturation random mutagenesis. The tHisA-D127X and tHisF-D130X variants with TrpF activity were identified by selection in vivo, and the proteins purified and characterized. The results obtained show that removal of the negatively charged carboxylate side-chain at the C-terminal end of beta-strand 5 is sufficient to establish TrpF activity in tHisA and tHisF, presumably because it allows the binding of the negatively charged TrpF substrate, phosphoribosylanthranilate. In contrast, the double mutants tHisA-D8N+D127V and tHisF-D11N+D130V did not show detectable activity, demonstrating that the aspartate residues at the C-terminal end of beta-strand 1 are essential for catalysis of the TrpF reaction. The ease with which TrpF activity can be established on both the tHisA and tHisF scaffolds supports the evolutionary relationship of these three enzymes and highlights the functional plasticity of the (betaalpha)(8)-barrel enzyme fold.     

Roles of active site tryptophans in substrate binding and catalysis by alpha-1,3 galactosyltransferase

Aromatic amino acids are frequent components of the carbohydrate binding sites of lectins and enzymes. Previous structural studies have shown that in alpha-1,3 galactosyltransferase, the binding site for disaccharide acceptor substrates is encircled by four tryptophans, residues 249, 250, 314, and 356. To investigate their roles in enzyme specificity and catalysis, we expressed and characterized variants of the catalytic domain of alpha-1,3 galactosyltransferase with substitutions for each tryptophan. Substitution of glycine for tryptophan 249, whose indole ring interacts with the nonpolar B face of glucose or GlcNAc, greatly increases the K(m) for the acceptor substrate. In contrast, the substitution of tyrosine for tryptophan 314, which interacts with the beta-galactosyl moiety of the acceptor and UDP-galactose, decreases k(cat) for the galactosyltransferase reaction but does not affect the low UDP-galactose hydrolase activity. Thus, this highly conserved residue stabilizes the transition state for the galactose transfer to disaccharide but not to water. High-resolution crystallographic structures of the Trp(249)Gly mutant and the Trp(314)Tyr mutant indicate that the mutations do not affect the overall structure of the enzyme or its interactions with ligands. Substitutions for tryptophan 250 have only small effects on catalytic activity, but mutation of tryptophan 356 to threonine reduces catalytic activity for both transferase and hydrolase activities and reduces affinity for the acceptor substrate. This residue is adjacent to the flexible C-terminus that becomes ordered on binding UDP to assemble the acceptor binding site and influence catalysis. The results highlight the diverse roles of these tryptophans in enzyme action and the importance of k(cat) changes in modulating glycosyltransferase specificity.     

Chemical and mutagenic investigations of fatty acid amide hydrolase: evidence for a family of serine hydrolases with distinct catalytic properties.

Fatty acid amide hydrolase (FAAH) is a membrane-bound enzyme responsible for the catabolism of neuromodulatory fatty acid amides, including anandamide and oleamide. FAAH's primary structure identifies this enzyme as a member of a diverse group of alkyl amidases, known collectively as the "amidase signature family". At present, this enzyme family's catalytic mechanism remains poorly understood. In this study, we investigated the catalytic features of FAAH through mutagenesis, affinity labeling, and steady-state kinetic methods. In particular, we focused on the respective roles of three serine residues that are conserved in all amidase signature enzymes (S217, S218, and S241 in FAAH). Mutation of each of these serines to alanine resulted in a FAAH enzyme bearing significant catalytic defects, with the S217A and S218A mutants showing 2300- and 95-fold reductions in k(cat), respectively, and the S241A mutant exhibiting no detectable catalytic activity. The double S217A:S218A FAAH mutant displayed a 230 000-fold decrease in k(cat), supporting independent catalytic functions for these serine residues. Affinity labeling of FAAH with a specific nucleophile reactive inhibitor, ethoxy oleoyl fluorophosphonate, identified S241 as the enzyme's catalytic nucleophile. The pH dependence of FAAH's k(cat) and k(cat)/K(m) implicated a base involved in catalysis with a pK(a) of 7.9. Interestingly, mutation of each of FAAH's conserved histidines (H184, H358, and H449) generated active enzymes, indicating that FAAH does not contain a Ser-His-Asp catalytic triad commonly found in other mammalian serine hydrolytic enzymes. The unusual properties of FAAH identified here suggest that this enzyme, and possibly the amidase signature family as a whole, may hydrolyze amides by a novel catalytic mechanism.     

Cleavage of various peptides with pitrilysin from Escherichia coli: kinetic analyses using beta-endorphin and its derivatives

Pitrilysin from Escherichia coli was overproduced, purified, and analyzed for enzymatic activity using 14 peptides as a substrate. Pitrilysin cleaved all the peptides, except for two of the smallest, at a limited number of sites, but showed little amino acid specificity. It cleaved beta-endorphin (beta-EP) most effectively, with a K(m) value of 0.36 microM and a k(cat) value of 750 min(-1). beta-EP consists of 31 residues and was predominantly cleaved by the enzyme at Lys(19)-Asn(20). Kinetic analyses using a series of beta-EP derivatives with N and/or C-terminal truncations and with amino acid substitutions revealed that three hydrophobic residues (Leu(14), Val(15), and Leu(17)) and the region 22-26 in beta-EP are responsible for high-affinity recognition by the enzyme. These two regions are located on the N- and C-terminal sides of the cleavage site in beta-EP, suggesting that the substrate binding pocket of pitrilysin spans its catalytic site.     

人双专一性磷酸酶活性位点Cys^124附近精氨酸突变及功能

为研究人双专一性磷酸酶活性位点Cys12 4 附近 3个带正电的精氨酸对酶催化功能的影响 ,用QuikChange定点突变方法获得 6个突变体 :R12 5L、R130 L、R130 K、R130 L/S131A、R158K和R158L。将含突变基因的重组质粒转化大肠杆菌菌株BL2 1(DE3) ,经IPTG诱导表达获得的目的蛋白质均以可溶形式存在。通过镍离子亲和层析纯化得到纯度大于 90 %的蛋白质。对人痘苗病毒H1相关磷酸酶 (VHR)及其突变体进行稳态动力学参数和竞争性抑制常数Ki 的测定 ,结果显示上述Arg130 和Arg158突变体的kcat/Km 值都较野生型有大幅度下降 ,而Ki 值有明显上升 ,表明 130和 15 8位的精氨酸是VHR活性所必需 ,而且可能与底物上带负电的磷酸基团结合有关。另外 ,单突变体R130 L和双突变体R130 L/S131A之间的kcat值相差很小 ,提示Arg130 单点突变后可能破坏了Ser131与Cys12 4 间的氢键。再者 ,R12 5L、R130 L和R158L突变体都降低了砷酸盐结合亲和性 ,暗示这 3个精氨酸残基侧链上的正电荷可能有助于底物与酶的结合。     

Streptococcus gordonii soluble inorganic pyrophosphatase: an important role for the interdomain region in enzyme activity

Streptococcus gordonii DL1(Challis) soluble inorganic pyrophosphatase was shown to be a homo dimer with a subunit molecular mass of 33407. In solution, in the presence of Mn(2+), the protein is ellipsoidal with an axial ratio of 3.37 and molecular mass of 67000. In the absence of the divalent cation, the molecular mass is unchanged but the axial ratio increases to 3.94. The enzyme, in the presence of 5 mM Mg(2+), at 25 degrees Celsius and pH 9.0, has K(m) and k(cat) values of 62 microM and 6290 s(-1), respectively. The free N- and C-terminal domains of Streptococcus gordonii PPase did not interact productively when mixed together. Replacing the interdomain region with that from Bacillus subtilis decreased the catalytic efficiency of the enzyme whereas inserting the same region from the Archaeglobus fulgidus thermophilic enzyme yielded an inactive protein. Substitution, deletion and insertion of amino acid residues in the interdomain region were found to affect the monomer dimer equilibrium in the absence of Mn(2+) ions. In the presence of these ions however the variant proteins were dimers. Proteins with altered interdomain regions also displayed a 2- to 625-fold decrease in catalytic efficiency. These data together with that of computer analysis show that the interdomain region has characteristics of a mechanical hinge. Modelling mutant proteins onto the wild type shows that the active site regions are not significantly perturbed. These results show that, although distant from the active site, the interdomain region plays a role in enzyme activity and both its length and composition are important. This supports the hypothesis that catalytic activity requires the N- and C terminal domains of the enzyme to open and close using the interdomain region as a hinge.