Site-directed mutagenesis of active-site-related residues in Torpedo acetylcholinesterase. Presence of a glutamic acid in the catalytic triad.

Site-directed mutagenesis was used to investigate the role of acidic amino acid residues close to the active site of Torpedo acetylcholinesterase. The recently determined atomic structure of this enzyme shows the conserved Glu-327, together with His-440 and Ser-200 as forming a catalytic triad, while the adjacent conserved Asp-326 points away from the active site. Transfection of appropriately mutated DNA into COS cells showed that the mutation of Asp-326----Asn had little effect on catalytic activity or the molecular forms expressed, suggesting no crucial structural or functional role for this residue. Mutation of Glu-327 to Gln or to Asp led to an inactive product. These results support the conclusions of the structural analysis for the two acidic residues.     

Identification of the enzymatic active site of CD38 by site-directed mutagenesis

CD38 is a ubiquitous protein originally identified as a lymphocyte antigen and recently also found to be a multifunctional enzyme participating in the synthesis and metabolism of two Ca(2+) messengers, cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate. It is homologous to Aplysia ADP-ribosyl cyclase, where the crystal structure has been determined. Residues of CD38 corresponding to those at the active site of the Aplysia cyclase were mutagenized. Changing Glu-226, which corresponded to the catalytic residue of the cyclase, to Asp, Asn, Gln, Leu, or Gly eliminated essentially all enzymatic activities of CD38, indicating it is most likely the catalytic residue. Photoaffinity labeling showed that E226G, nevertheless, retained substantial NAD binding activity. The secondary structures of these inactive mutants as measured by circular dichroism were essentially unperturbed as compared with the wild type. Other nearby residues were also investigated. The mutants D147V and E146L showed 7- and 19-fold reduction in NADase activity, respectively. The cADPR hydrolase activity of the two mutants was similarly reduced. Asp-155, on the other hand, was crucial for the GDP-ribosyl cyclase activity since its substitution with either Glu, Asn, or Gln stimulated the activity 3-15-fold, whereas other activities remained essentially unchanged. In addition to these acidic residues, two tryptophans were also important, since all enzyme activities of W125F, W125Y, W189G and W189Y were substantially reduced. This is consistent with the two tryptophans serving a substrate positioning function. A good correlation was observed when the NADase activity of all the mutants was plotted against the cADPR hydrolase activity. Homology modeling revealed all these critical residues are clustered in a pocket near the center of the CD38 molecule. The results indicate a strong structural homology between the active sites of CD38 and the Aplysia cyclase.     

Human lysosomal alpha-glucosidase. Characterization of the catalytic site.

The substrate analogue conduritol B epoxide (CBE) is demonstrated to be an active site-directed inhibitor of human lysosomal alpha-glucosidase. A competitive mode of inhibition is obtained with glycogen as natural and 4-methylumbelliferyl-alpha-D-glucopyranoside as artificial substrate. The inactivation of the enzyme is time and concentration dependent and results in the covalent binding of CBE. Catalytic activity is required for binding to occur. CBE-labeled peptides containing the catalytic residue of lysosomal alpha-glucosidase were isolated and identified by microsequencing and amino acid analysis. The peptides appeared to originate from a protein domain which is highly conserved among alpha-amylases, maltase, glucoamylases, and transglucanosylases. Based on the sequence similarity and the mechanism of CBE binding, Asp-518 is predicted to be the essential carboxylate in the active site of lysosomal alpha-glucosidase. The functional importance of Asp-518 and other residues around the catalytic site was studied by expression of in vitro mutagenized alpha-glucosidase cDNA in transiently transfected COS cells. Substitution of Asp-513 by Glu-513 is shown to interfere with the posttranslational modification and the intracellular transport of the alpha-glucosidase precursor. The residues Trp-516 and Asp-518 are demonstrated to be critical for catalytic function.     

Identification of amino acid residues essential for the substrate specificity of Flavobacterium sp. endo-beta-N-acetylglucosaminidase

The gene encoding the endo-beta-N-acetylglucosaminidase from Flavobacterium sp. (Endo-Fsp) was sequenced. The Endo-Fsp gene was overexpressed in Escherichia coli cells, and was purified from inclusion bodies after denaturation by 8 M urea. The renatured Endo-Fsp had the same optimum pH and substrate specificity as the native enzyme. Endo-Fsp had 60% sequence identity with the endo-beta-N-acetylglucosaminidase from Streptomyces plicatus (Endo-H), and the putative catalytic residues were conserved. Site-directed mutagenesis was done at conserved residues based on the three-dimensional structure and mutagenesis of Endo-H. The mutant of Glu-128, corresponding to Glu-132 in Endo-H and identified as an active site residue, was inactivated. Mutagenesis around the predicted active site of Endo-Fsp reduced the enzymatic activity. Moreover, the hydrolytic activity toward hybrid-type oligosaccharides was decreased compared to that toward high-mannose type oligosaccharides by mutagenesis of Asp-126 and Asp-127. Therefore, site-directed mutagenesis of some of these conserved residues indicates that the predicted active sites are essential to the enzymatic activity of Endo-Fsp, and may have similar roles in catalysis as their counterparts in Endo-H.     

A fungal cellulase shows sequence homology with the active site of hen egg-white lysozyme

The N-terminal amino acid sequence of an endo-β-1,4-glucanase from the cellulase complex of the white-rot fungus Schizophyllum commune has been determined. The sequence from Glu-33 to Tyr-51 was homologous with the active site sequences of various hen egg-white type lysozymes, including lysozyme catalytic residues (Glu-35, Asp-52) and substrate binding residue Asn-44. The homology offers evidence for a lysozyme-type mechanism in enzymic hydrolysis of cellulose.     

Functional mapping of charged residues of the 82-116 sequence in factor Xa: evidence that lysine 96 is a factor Va independent recognition site for prothrombin in the prothrombinase complex

It has been hypothesized that two antiparallel structures comprised of residues 82-91 and 102-116 in factor Xa (fXa) may harbor a factor Va- (fVa-) dependent prothrombin recognition site in the prothrombinase complex. There are 11 charged residues in the 82-116 loop of human fXa (Glu-84, Glu-86, Lys-90, Arg-93, Lys-96, Glu-97, Asp-100, Asp-102, Arg-107, Lys-109, and Arg-115). With the exception of Glu-84, which did not express, and Asp-102, which is a catalytic residue, we expressed the Ala substitution mutants of all other residues and evaluated their proteolytic and amidolytic activities in both the absence and presence of fVa. K96A and K109A activated prothrombin with 5-10-fold impaired catalytic efficiency in the absence of fVa. All mutants, however, exhibited normal activity toward the substrate in the presence of fVa. K109A also exhibited impaired amidolytic activity and affinity for Na(+); however, both fVa and higher Na(+) restored the catalytic defect caused by the mutation. Analysis of the X-ray crystal structure of fXa indicated that Glu-84 may interact by a salt bridge with Lys-109, explaining the lack of expression of E84A and the lower activity of K109A in the absence of fVa. These results suggest that none of the residues under study is a fVa-dependent recognition site for prothrombin in the prothrombinase complex; however, Lys-96 is a recognition site for the substrate independent of the cofactor. Moreover, the 82-116 loop is energetically linked to fVa and Na(+) binding sites of the protease.     

Molecular dissection of Na+ binding to thrombin

Na(+) binding near the primary specificity pocket of thrombin promotes the procoagulant, prothrombotic, and signaling functions of the enzyme. The effect is mediated allosterically by a communication between the Na(+) site and regions involved in substrate recognition. Using a panel of 78 Ala mutants of thrombin, we have mapped the allosteric core of residues that are energetically linked to Na(+) binding. These residues are Asp-189, Glu-217, Asp-222, and Tyr-225, all in close proximity to the bound Na(+). Among these residues, Asp-189 shares with Asp-221 the important function of transducing Na(+) binding into enhanced catalytic activity. None of the residues of exosite I, exosite II, or the 60-loop plays a significant role in Na(+) binding and allosteric transduction. X-ray crystal structures of the Na(+)-free (slow) and Na(+)-bound (fast) forms of thrombin, free or bound to the active site inhibitor H-d-Phe-Pro-Arg-chloromethyl-ketone, document the conformational changes induced by Na(+) binding. The slow --> fast transition results in formation of the Arg-187:Asp-222 ion pair, optimal orientation of Asp-189 and Ser-195 for substrate binding, and a significant shift of the side chain of Glu-192 linked to a rearrangement of the network of water molecules that connect the bound Na(+) to Ser-195 in the active site. The changes in the water network and the allosteric core explain the thermodynamic signatures linked to Na(+) binding and the mechanism of thrombin activation by Na(+). The role of the water network uncovered in this study establishes a new paradigm for the allosteric regulation of thrombin and other Na(+)-activated enzymes involved in blood coagulation and the immune response.     

Interaction of catalytic-site mutants of Bacillus subtilis alpha-amylase with substrates and acarbose.

The interactions of the three catalytic-site mutants of Bacillus subtilis alpha-amylase/(DN176 [Asp-176----Asn], EQ208 [Glu-208----Gln] and DN269 [Asp-269----Asn]) with substrates and a pseudo-oligosaccharide inhibitor, acarbose, have been studied by means of difference absorption spectroscopy and affinity chromatography. The addition of maltopentaose or soluble starch to the inactive mutant enzymes mostly resulted in difference spectra characteristic of tryptophan perturbation, enabling determination of the dissociation constants. The results show that conversion of Glu-208 to Gln greatly enhanced substrate binding, implying that Glu-208 interacts unfavorably with the substrate's ground state, preventing its optimal fit to the active site. The affinity for acarbose was greatly reduced in DN269 and EQ208, but less so in DN176, suggesting that Asp-269 and Glu-208 are more important than Asp-176 in stabilizing the transition state. These results are consistent with Glu-208 and Asp-269 being the key catalytic residues, as proposed for Taka-amylase A.     

Glutamate 83 is important for stabilization of domain-domain conformation of Thermus aquaticus glycerol kinase

The gene glpK, encoding glycerol kinase (GlpK) of Thermus aquaticus, has recently been identified. The protein encoded by glpK was found to have an unusually high identity of 81% with the sequence of GlpK from Bacillus subtilis. Three residues (Arg-82, Glu-83, and Asp-244) of T. aquaticus GlpK are conserved in all the known GlpK sequences, including those from various bacteria, yeast and human. The roles that these three residues play in the catalytic mechanism were investigated by using site-directed mutagenesis to produce three mutants: Arg-82-Ala, Glu-83-Ala, and Asp-244-Ala. Replacement of Asp-244 by Ala resulted in a complete loss of activity, thus suggesting that Asp-244 is important for catalysis. Taking k(cat)/K(m) as a simple measure of catalytic efficiency, the mutants Arg-82-Ala and Glu-83-Ala were judged to cause 190- and 37,000-fold decrease, respectively, when compared to the wild-type GlpK. Thus, these three residues play a critical role in the catalytic mechanism. However, only mutant Glu-83-Ala was cleaved by alpha-chymotrypsin, and proteolysis studies showed that the mutant Glu-83-Ala involves a change in the exposure of Tyr-331 at the alpha-chymotrypsin site. This indicates a large domain conformational change, since the residues corresponding to Glu-83 and Tyr-331 in the Escherichia coli GlpK sequence are located in domain IB and domain IIB, respectively. The apparent conformational change caused by replacement of Glu-83 leads us to propose that Glu-83 is an important residue for stabilization of domain conformation.     

An essential role of Glu-243 and His-239 in the phosphotransfer reaction catalyzed by pyruvate dehydrogenase kinase

This study was undertaken to examine the mechanistic significance of two highly conserved residues positioned in the active site of pyruvate dehydrogenase kinase, Glu-243 and His-239. We used site-directed mutagenesis to convert Glu-243 to Ala, Asp, or Gln and His-239 to Ala. The resulting mutant kinases demonstrated a greatly reduced capacity for phosphorylation of pyruvate dehydrogenase. The Glu-243 to Asp mutant had approximately 2% residual activity, whereas the Glu-243 to Ala or Gln mutants exhibited less than 0.5 and 0.1% residual activity, respectively. Activity of the His-239 to Ala mutant was decreased by approximately 90%. Active-site titration with [alpha-(32)P]ATP revealed that neither Glu-243 nor His-239 mutations affected nucleotide binding. All mutant kinases showed similar or even somewhat greater affinity than the wild-type kinase toward the protein substrate, pyruvate dehydrogenase complex. Furthermore, neither of the mutations affected the inter-subunit interactions. Finally, pyruvate dehydrogenase kinase was found to possess a weak ATP hydrolytic activity, which required Glu-243 and His-239 similar to the kinase activity. Based on these observations, we propose a mechanism according to which the invariant glutamate residue (Glu-243) acts as a general base catalyst, which activates the hydroxyl group on a serine residue of the protein substrate for direct attack on the gamma phosphate. The glutamate residue in turn might be further polarized through interaction with the neighboring histidine residue (His-239).     

Identification of catalytic residues of Ca2+-independent 1,2-alpha-D-mannosidase from Aspergillus saitoi by site-directed mutagenesis

The roles of six conserved active carboxylic acids in the catalytic mechanism of Aspergillus saitoi 1,2-alpha-d-mannosidase were studied by site-directed mutagenesis and kinetic analyses. We estimate that Glu-124 is a catalytic residue based on the drastic decrease of kcat values of the E124Q and E124D mutant enzyme. Glu-124 may work as an acid catalyst, since the pH dependence of its mutants affected the basic limb. D269N and E411Q were catalytically inactive, while D269E and E411D showed considerable activity. This indicated that the negative charges at these points are essential for the enzymatic activity and that none of these residues can be a base catalyst in the normal sense. Km values of E273D, E414D, and E474D mutants were greatly increased to 17-31-fold wild type enzyme, and the kcat values were decreased, suggesting that each of them is a binding site of the substrate. Ca2+, essential for the mammalian and yeast enzymes, is not required for the enzymatic activity of A. saitoi 1,2-alpha-d-mannosidase. EDTA inhibits the Ca2+-free 1,2-alpha-d-mannosidase as a competitive inhibitor, not as a chelator. We deduce that the Glu-124 residue of A. saitoi 1,2-alpha-d-mannosidase is directly involved in the catalytic mechanism as an acid catalyst, whereas no usual catalytic base is directly involved. Ca2+ is not essential for the activity. The catalytic mechanism of 1,2-alpha-d-mannosidase may deviate from that typical glycosyl hydrolase.     

Mutational, kinetic, and NMR studies of the roles of conserved glutamate residues and of lysine-39 in the mechanism of the MutT pyrophosphohydrolase

The MutT enzyme catalyzes the hydrolysis of nucleoside triphosphates (NTP) to NMP and PP(i) by nucleophilic substitution at the rarely attacked beta-phosphorus. The solution structure of the quaternary E-M(2+)-AMPCPP-M(2+) complex indicated that conserved residues Glu-53, -56, -57, and -98 are at the active site near the bound divalent cation possibly serving as metal ligands, Lys-39 is positioned to promote departure of the NMP leaving group, and Glu-44 precedes helix I (residues 47-59) possibly stabilizing this helix which contributes four catalytic residues to the active site [Lin, J. , Abeygunawardana, C., Frick, D. N., Bessman, M. J., and Mildvan, A. S. (1997) Biochemistry 36, 1199-1211]. To test these proposed roles, the effects of mutations of each of these residues on the kinetic parameters and on the Mn(2+), Mg(2+), and substrate binding properties were examined. The largest decreases in k(cat) for the Mg(2+)-activated enzyme of 10(4.7)- and 10(2.6)-fold were observed for the E53Q and E53D mutants, respectively, while 97-, 48-, 25-, and 14-fold decreases were observed for the E44D, E56D, E56Q, and E44Q mutations, respectively. Smaller effects on k(cat) were observed for mutations of Glu-98 and Lys-39. For wild type MutT and its E53D and E44D mutants, plots of log(k(cat)) versus pH exhibited a limiting slope of 1 on the ascending limb and then a hump, i.e., a sharply defined maximum near pH 8 followed by a plateau, yielding apparent pK(a) values of 7.6 +/- 0.3 and 8.4 +/- 0.4 for an essential base and a nonessential acid catalyst, respectively, in the active quaternary MutT-Mg(2+)-dGTP-Mg(2+) complex. The pK(a) of 7.6 is assigned to Glu-53, functioning as a base catalyst in the active quaternary complex, on the basis of the disappearance of the ascending limb of the pH-rate profile of the E53Q mutant, and its restoration in the E53D mutant with a 10(1.9)-fold increase in (k(cat))(max). The pK(a) of 8.4 is assigned to Lys-39 on the basis of the disappearance of the descending limb of the pH-rate profile of the K39Q mutant, and the observation that removal of the positive charge of Lys-39, by either deprotonation or mutation, results in the same 8.7-fold decrease in k(cat). Values of k(cat) of both wild type MutT and the E53Q mutant were independent of solvent viscosity, indicating that a chemical step is likely to be rate-limiting with both. A liganding role for Glu-53 and Glu-56, but not Glu-98, in the binary E-M(2+) complex is indicated by the observation that the E53Q, E53D, E56Q, and E56D mutants bound Mn(2+) at the active site 36-, 27-, 4.7-, and 1.9-fold weaker, and exhibited 2.10-, 1.50-, 1.12-, and 1.24-fold lower enhanced paramagnetic effects of Mn(2+), respectively, than the wild type enzyme as detected by 1/T(1) values of water protons, consistent with the loss of a metal ligand. However, the K(m) values of Mg(2+) and Mn(2+) indicate that Glu-56, and to a lesser degree Glu-98, contribute to metal binding in the active quaternary complex. Mutations of the more distant but conserved residue Glu-44 had little effect on metal binding or enhancement factors in the binary E-M(2+) complexes. Two-dimensional (1)H-(15)N HSQC and three-dimensional (1)H-(15)N NOESY-HSQC spectra of the kinetically damaged E53Q and E56Q mutants showed largely intact proteins with structural changes near the mutated residues. Structural changes in the kinetically more damaged E44D mutant detected in (1)H-(15)N HSQC spectra were largely limited to the loop I-helix I motif, suggesting that Glu-44 stabilizes the active site region. (1)H-(15)N HSQC titrations of the E53Q, E56Q, and E44D mutants with dGTP showed changes in chemical shifts of residues lining the active site cleft, and revealed tighter nucleotide binding by these mutants, indicating an intact substrate binding site. (ABSTRACT TRUNCATED)     

Homology modelling and site-directed mutagenesis studies of the epoxide hydrolase from Phanerochaete chrysosporium

Three-dimensional structural model of epoxide hydrolase (PchEHA) from Phanerochaete chrysosporium was constructed based on X-ray structure of Agrobacterium radiobacter AD1 sEH using SWISS-MODEL server. Conserved residues constituting the active site cavity were identified, of which the functional roles of 14 residues were determined by site-directed mutagenesis. In catalytic triad, Asp105 and His308 play a leading role in alkylation and hydrolysis steps, respectively. Distance between Asp105 and epoxide ring of substrate may determine the regiospecificity in the substrate docking model. Asp277 located at the entrance of substrate tunnel is concerned with catalysis but not essential. D307E had the highest activity and lower enantioselectivity among 14 mutants, suggesting Asp307 may be involved in choice of substrate configuration. Y159F and Y241F almost exhibited no activity, indicating that they are essential to bind substrate and facilitate opening of epoxide ring. Besides, His35-Gly36-Asn37-Pro38, Trp106 and Trp309 surrounding Asp105, may coordinate the integration of active site cavity and influence substrate binding. Especially, W106I reversed the enantioselectivity, perhaps due to more deteriorative impact on the preferred (R)-styrene oxide. Gly65 and Gly67 occurring at β-turns and Gly36 are vital in holding protein conformation. Conclusively, single conserved residue around the active sites has an important impact on catalytic properties.     

Biochemical characterization of the chondroitinase B active site

Chondroitinase B from Flavobacterium heparinum is the only known lyase that cleaves the glycosaminoglycan, dermatan sulfate (DS), as its sole substrate. A recent co-crystal structure of chondroitinase B with a disaccharide product of DS depolymerization has provided some insight into the location of the active site and suggested potential roles of some active site residues in substrate binding and catalysis. However, this co-crystal structure was not representative of the actual enzyme-substrate complex, because the disaccharide product did not have the right length or the chemical structure of the minimal substrate (tetrasaccharide) involved in catalysis. Therefore, only a limited picture of the functional role of active site residues in DS depolymerization was presented in previous structural studies. In this study, by docking a DS tetrasaccharide into the proposed active site of the enzyme, we have identified novel roles of specific active site amino acids in the catalytic function of chondroitinase B. Our conformational analysis also revealed a unique, symmetrical arrangement of active site amino acids that may impinge on the catalytic mechanism of action of chondroitinase B. The catalytic residues Lys-250, Arg-271, His-272, and Glu-333 along with the substrate binding residues Arg-363 and Arg-364 were mutated using site-directed mutagenesis, and the kinetics and product profile of each mutant were compared with recombinant chondroitinase B. Mutating Lys-250 to alanine resulted in inactivation of the enzyme, potentially attributable to the role of the residue in stabilizing the carbanion intermediate formed during enzymatic catalysis. The His-272 and Glu-333 mutants showed diminished enzymatic activity that could be indicative of a possible role for one or both residues in the abstraction of the C-5 proton from the galactosamine. In addition, the Arg-364 mutant had an altered product profile after exhaustive digestion of DS, suggesting a role for this residue in defining the substrate specificity of chondroitinase B.     

Mutations of endo-beta-N-acetylglucosaminidase H active site residueAs sp130 anG glu132: activities and conformations

Endo-beta-N-acetylglucosaminidase H hydrolyzes the beta-(1-4)-glycosidic link of the N,N'-diacetylchitobiose core of high-mannose and hybrid asparagine-linked oligosaccharides. Seven mutants of the active site residues, Asp130 and Glu132, have been prepared, assayed, and crystallized. They include single site mutants of each residue to the corresponding amide, to Ala and to the alternate acidic residue, and to the double amide mutant. The mutants of Asp130 are more active than the corresponding Glu132 mutants, consistent with the assignment of the latter residue as the primary catalytic residue. The amide mutants are more active than the alternate acidic residue mutants, which in turn are more active than the Ala mutants. The structures of the Asn mutant of Asp130 and the double mutant are very similar to that of the wild-type enzyme. Several residues surrounding the mutated residues, including some that form part of the core of the beta-barrel and especially Tyr168 and Tyr244, adopt a very different conformation in the structures of the other two mutants of Asp130 and in the Asp mutant of Glu132. The results show that the residues in the upper layers of the beta-barrel can organize into two very distinct packing arrangements that depend on subtle electrostatic and steric differences and that greatly affect the geometry of the substrate-binding cleft. Consequently, the relative activities of several of the mutants are defined by structural changes, leading to impaired substrate binding, in addition to changes in functionality.     

Structural Analysis of a Family 101 Glycoside Hydrolase in Complex with Carbohydrates Reveals Insights into Its Mechanism

O-Linked glycosylation is one of the most abundant post-translational modifications of proteins. Within the secretory pathway of higher eukaryotes, the core of these glycans is frequently an N-acetylgalactosamine residue that is α-linked to serine or threonine residues. Glycoside hydrolases in family 101 are presently the only known enzymes to be able to hydrolyze this glycosidic linkage. Here we determine the high-resolution structures of the catalytic domain comprising a fragment of GH101 from Streptococcus pneumoniae TIGR4, SpGH101, in the absence of carbohydrate, and in complex with reaction products, inhibitor, and substrate analogues. Upon substrate binding, a tryptophan lid (residues 724-WNW-726) closes on the substrate. The closing of this lid fully engages the substrate in the active site with Asp-764 positioned directly beneath C1 of the sugar residue bound within the −1 subsite, consistent with its proposed role as the catalytic nucleophile. In all of the bound forms of the enzyme, however, the proposed catalytic acid/base residue was found to be too distant from the glycosidic oxygen (>4.3 Å) to serve directly as a general catalytic acid/base residue and thereby facilitate cleavage of the glycosidic bond. These same complexes, however, revealed a structurally conserved water molecule positioned between the catalytic acid/base and the glycosidic oxygen. On the basis of these structural observations we propose a new variation of the retaining glycoside hydrolase mechanism wherein the intervening water molecule enables a Grotthuss proton shuttle between Glu-796 and the glycosidic oxygen, permitting this residue to serve as the general acid/base catalytic residue.     

M42 aminopeptidase catalytic site: the structural and functional role of a strictly conserved aspartate residue

The M42 aminopeptidases are a family of dinuclear aminopeptidases widely distributed in Prokaryotes. They are potentially associated to the proteasome, achieving complete peptide destruction. Their most peculiar characteristic is their quaternary structure, a tetrahedron-shaped particle made of twelve subunits. The catalytic site of M42 aminopeptidases is defined by seven conserved residues. Five of them are involved in metal ion binding which is important to maintain both the activity and the oligomeric state. The sixth conserved residue, a glutamate, is the catalytic base deprotonating the water molecule during peptide bond hydrolysis. The seventh residue is an aspartate whose function remains poorly understood. This aspartate residue, however, must have a critical role as it is strictly conserved in all MH clan enzymes. It forms some kind of catalytic triad with the histidine residue and the metal ion of the M2 binding site. We assess its role in TmPep1050, an M42 aminopeptidase of Thermotoga maritima, through a mutational approach. Asp-62 was substituted with alanine, asparagine, or glutamate residue. The Asp-62 substitutions completely abolished TmPep1050 activity and impeded dodecamer formation. They also interfered with metal ion binding as only one cobalt ion is bound per subunit instead of two. The structure of Asp62Ala variant was solved at 1.5 Å showing how the substitution has an impact on the active site fold. We propose a structural role for Asp-62, helping to stabilize a crucial loop in the active site and to position correctly the catalytic base and a metal ion ligand of the M1 site.     

Structural analysis of the zinc hydroxide–Thr-199–Glu-106 hydrogen-bond network in human carbonic anhydrase II

The singnificance of the zinc hydroxide–Thr-199–Glu-106 hydrogen-bond network in the active site of human carbonic anhydrase II has been examined by X-ray crystallographic analyses of site-specific mutants. Mutants with Ala-199 and Ala-106 or Gln-106 have low catalytic activities, while a mutant with Asp-106 has almost full CO₂ hydration activity. The structures of these four mutants, as well as that of the bicarbonate complex of the mutant with Ala-199, have been determined at 1.7 to 2.2 Å resolution. Removal of the γ atoms of residue 199 leads to distorted tetrahedral geometry at the zine ion, and a catalytically important zinc-bound water molecule has moved towards Glu-106. In the bicarbonate complex of the mutant with Ala-199 one oxygen atom from bicarbonate binds to zinc without displacing this water molecule. Tetrahedral coordination geometries are retained in the mutants at position 106. The mutants with Ala-106 and Gln-106 have a zinc-bound sulfate ion, whereas this sulfate site is only partially occupied in the mutant with Asp-106. The hydrogen-bond network seems to be “reversed” in the mutants with Ala-106 and Gln-106. The network is preserved as in native enzyme in the mutant with Asp-106 but the side chain of Asp-106 is more extended than that of Glu-106 in the native enzyme. These results illustrate the importance of Glu-106 and Thr-199 for controlling the precise coordination geometry of the zinc ion and its ligand preferences with results in an optimal orientation of a zine-bound hydroxide ion for an attack on the CO₂ substrate. © 1993 Wiley-Liss, Inc.