A rationalization of the acidic pH dependence for stromelysin-1 (Matrix metalloproteinase-3) catalysis and inhibition

The pH dependence of matrix metalloproteinase (MMP) catalysis is described by a broad bell-shaped curve, indicating the involvement of two unspecified ionizable groups in proteolysis. Stromelysin-1 has a third pK(a) near 6, resulting in a uniquely sharp acidic catalytic optimum, which has recently been attributed to His(224). This suggests the presence of a critical, but unidentified, S1' substructure. Integrating biochemical characterizations of inhibitor-enzyme interactions with active site topography from corresponding crystal structures, we isolated contributions to the pH dependence of catalysis and inhibition of active site residues Glu(202) and His(224). The acidic pK(a) 5.6 is attributed to the Glu(202).zinc.H(2)O complex, consistent with a role for the invariant active site Glu as a general base in MMP catalysis. The His(224)-dependent substructure is identified as a tripeptide (Pro(221)-Leu(222)-Tyr(223)) that forms the substrate cleft lower wall. Substrate binding induces a beta-conformation in this sequence, which extends and anchors the larger beta-sheet of the enzyme. substrate complex and appears to be essential for productive substrate binding. Because the PXY tripeptide is strictly conserved among MMPs, this "beta-anchor" may represent a common motif required for macromolecular substrate hydrolysis. The striking acidic profile of stromelysin-1 defined by the combined ionization of Glu(202) and His(224) allows the design of highly selective inhibitors.     

Role of two active site Glu residues in the molecular action of botulinum neurotoxin endopeptidase

Botulinum neurotoxin type A (BoNT/A) light chain (LC) is a zinc endopeptidase that causes neuroparalysis by blocking neurotransmitter release at the neuromuscular junctions. The X-ray crystal structure of the toxin reveals that His223 and His227 of the Zn(2+) binding motif HEXXH directly coordinate the active site zinc. Two Glu residues (Glu224 and Glu262) are also part of the active site, with Glu224 coordinating the zinc via a water molecule whereas Glu262 coordinates the zinc directly as the fourth ligand. In the past we have investigated the topographical role of Glu224 by replacing it with Asp thus reducing the side chain length by 1.4 A that reduced the endopeptidase activity dramatically [L. Li, T. Binz, H. Niemann, and B.R. Singh, Probing the role of glutamate residue in the zinc-binding motif of type A botulinum neurotoxin light chain, Biochemistry 39 (2000) 2399-2405]. In this study we have moved the Glu 224 laterally by a residue (HXEXH) to assess its positional influence on the endopeptidase activity, which was completely lost. The functional implication of Glu262 was investigated by replacing this residue with aspartate and glutamine using site-directed mutagenesis. Substitution of Glu262 with Asp resulted in a 3-fold decrease in catalytic efficiency. This mutation did not induce any significant structural alterations in the active site and did not interfere with substrate binding. Substitution of Glu262 with Gln however, dramatically impaired the enzymatic activity and this is accompanied by global alterations in the active site conformation in terms of topography of aromatic amino acid residues, zinc binding, and substrate binding, resulting from the weakened interaction between the active site zinc and Gln. These results suggest a pivotal role of the negatively charged carboxyl group of Glu262 which may play a critical role in enhancing the stability of the active site with strong interaction with zinc. The zinc may thus play structural role in addition to its catalytic role.     

Identification of metal ligands in the Clostridium histolyticum ColH collagenase

A Clostridium histolyticum 116-kDa collagenase has an H415EXXH motif but not the third zinc ligand, as found in already characterized zinc metalloproteinases. To identify its catalytic site, we mutated the codons corresponding to the three conserved residues in the motif to other amino acid residues. The mutation affecting His415 or His419 abolished catalytic activity and zinc binding, while that affecting Glu416 did the former but not the latter. These results suggest that the motif forms the catalytic site. We also mutated the codons corresponding to other amino acid residues that are likely zinc ligands. The mutation affecting Glu447 decreased markedly both the enzymatic activity and the zinc content, while that affecting Glu446 or Glu451 had smaller effects on activity and zinc binding. These mutations caused a decrease in kcat but no significant change in Km. These results are consistent with the hypothesis that Glu447 is the third zinc ligand. The spacing of the three zinc ligands is the same in all known clostridial collagenases but not in other known gluzincins, indicating that they form a new gluzincin subfamily. The effects of mutations affecting Glu446 and Glu451 suggest that the two residues are also involved in catalysis, possibly through an interaction with the two zinc-binding histidine residues.     

Biosynthesis of pteridines. Reaction mechanism of GTP cyclohydrolase I

GTP cyclohydrolase I catalyses the hydrolytic release of formate from GTP followed by cyclization to dihydroneopterin triphosphate. The enzymes from bacteria and animals are homodecamers containing one zinc ion per subunit. Replacement of Cys110, Cys181, His112 or His113 of the enzyme from Escherichia coli by serine affords catalytically inactive mutant proteins with reduced capacity to bind zinc. These mutant proteins are unable to convert GTP or the committed reaction intermediate, 2-amino-5-formylamino-6-(beta-ribosylamino)-4(3H)-pyrimidinone 5'-triphosphate, to dihydroneopterin triphosphate. The crystal structures of GTP complexes of the His113Ser, His112Ser and Cys181Ser mutant proteins determined at resolutions of 2.5A, 2.8A and 3.2A, respectively, revealed the conformation of substrate GTP in the active site cavity. The carboxylic group of the highly conserved residue Glu152 anchors the substrate GTP, by hydrogen bonding to N-3 and to the position 2 amino group. Several basic amino acid residues interact with the triphosphate moiety of the substrate. The structure of the His112Ser mutant in complex with an undefined mixture of nucleotides determined at a resolution of 2.1A afforded additional details of the peptide folding. Comparison between the wild-type and mutant enzyme structures indicates that the catalytically active zinc ion is directly coordinated to Cys110, Cys181 and His113. Moreover, the zinc ion is complexed to a water molecule, which is in close hydrogen bond contact to His112. In close analogy to zinc proteases, the zinc-coordinated water molecule is suggested to attack C-8 of the substrate affording a zinc-bound 8R hydrate of GTP. Opening of the hydrated imidazole ring affords a formamide derivative, which remains coordinated to zinc. The subsequent hydrolysis of the formamide motif has an absolute requirement for zinc ion catalysis. The hydrolysis of the formamide bond shows close mechanistic similarity with peptide hydrolysis by zinc proteases.     

Crystal structure of D-aminoacylase from Alcaligenes faecalis DA1. A novel subset of amidohydrolases and insights into the enzyme mechanism

D-Aminoacylase is an attractive candidate for commercial production of D-amino acids through its catalysis in the hydrolysis of N-acyl-D-amino acids. We report here the first D-aminoacylase crystal structure from A. faecalis at 1.5-A resolution. The protein comprises a small beta-barrel, and a catalytic (betaalpha)(8)-barrel with a 63-residue insertion. The enzyme structure shares significant similarity to the alpha/beta-barrel amidohydrolase superfamily, in which the beta-strands in both barrels superimpose well. Unexpectedly, the enzyme binds two zinc ions with widely different affinities, although only the tightly bound zinc ion is required for activity. One zinc ion is coordinated by Cys(96), His(220), and His(250), while the other is loosely chelated by His(67), His(69), and Cys(96). This is the first example of the metal ion coordination by a cysteine residue in the superfamily. Therefore, D-aminoacylase defines a novel subset and is a mononuclear zinc metalloenzyme but containing a binuclear active site. The preferred substrate was modeled into a hydrophobic pocket, revealing the substrate specificity and enzyme catalysis. The 63-residue insertion containing substrate-interacting residues may act as a gate controlling access to the active site, revealing that the substrate binding would induce a closed conformation to sequester the catalysis from solvent.     

Heparan/chondroitin sulfate biosynthesis. Structure and mechanism of human glucuronyltransferase I

Human beta1,3-glucuronyltransferase I (GlcAT-I) is a central enzyme in the initial steps of proteoglycan synthesis. GlcAT-I transfers a glucuronic acid moiety from the uridine diphosphate-glucuronic acid (UDP-GlcUA) to the common linkage region trisaccharide Gal beta 1-3Gal beta 1-4Xyl covalently bound to a Ser residue at the glycosaminylglycan attachment site of proteoglycans. We have now determined the crystal structure of GlcAT-1 at 2.3 A in the presence of the donor substrate product UDP, the catalytic Mn(2+) ion, and the acceptor substrate analog Gal beta 1-3Gal beta 1-4Xyl. The enzyme is a alpha/beta protein with two subdomains that constitute the donor and acceptor substrate binding site. The active site residues lie in a cleft extending across both subdomains in which the trisaccharide molecule is oriented perpendicular to the UDP. Residues Glu(227), Asp(252), and Glu(281) dictate the binding orientation of the terminal Gal-2 moiety. Residue Glu(281) is in position to function as a catalytic base by deprotonating the incoming 3-hydroxyl group of the acceptor. The conserved DXD motif (Asp(194), Asp(195), Asp(196)) has direct interaction with the ribose of the UDP molecule as well as with the Mn(2+) ion. The key residues involved in substrate binding and catalysis are conserved in the glucuronyltransferase family as well as other glycosyltransferases.     

Expression and characterization of the biofilm-related and carnosine-hydrolyzing aminoacylhistidine dipeptidase from Vibrio alginolyticus

The biofilm-related and carnosine-hydrolyzing aminoacylhistidine dipeptidase (pepD) gene from Vibrio alginolyticus was cloned and sequenced. The recombinant PepD protein was produced and biochemically characterized and the putative active-site residues responsible for metal binding and catalysis were identified. The recombinant enzyme, which was identified as a homodimeric dipeptidase in solution, exhibited broad substrate specificity for Xaa-His and His-Xaa dipeptides, with the highest activity for the His-His dipeptide. Sequence and structural homologies suggest that the enzyme is a member of the metal-dependent metallopeptidase family. Indeed, the purified enzyme contains two zinc ions per monomer. Reconstitution of His.Tag-cleaved native apo-PepD with various metal ions indicated that enzymatic activity could be optimally restored when Zn2+ was replaced with other divalent metal ions, including Mn2+, Co2+, Ni2+, Cu2+ and Cd2+, and partially restored when Zn2+ was replaced with Mg2+. Structural homology modeling of PepD also revealed a 'catalytic domain' and a 'lid domain' similar to those of the Lactobacillus delbrueckii PepV protein. Mutational analysis of the putative active-site residues supported the involvement of His80, Asp119, Glu150, Asp173 and His461 in metal binding and Asp82 and Glu149 in catalysis. In addition, individual substitution of Glu149 and Glu150 with aspartic acid resulted in the partial retention of enzymatic activity, indicating a functional role for these residues on the catalysis and zinc ions, respectively. These effects may be necessary either for the activation of the catalytic water molecule or for the stabilization of the substrate-enzyme tetrahedral intermediate. Taken together, these results may facilitate the design of PepD inhibitors for application in antimicrobial treatment and antibody-directed enzyme prodrug therapy.     

Identification of critical residues in the active site of porcine membrane-bound aminopeptidase P

The membrane-bound form of mammalian aminopeptidase P (AP-P; EC 3.4. 11.9) is a mono-zinc-containing enzyme that lacks any of the typical metal binding motifs found in other zinc metalloproteases. To identify residues involved in metal binding and catalysis, sequence and structural information was used to align the sequence of porcine membrane-bound AP-P with other members of the peptidase clan MG, including Escherichia coli AP-P and methionyl aminopeptidases. Residues predicted to be critical for activity were mutated and the resultant proteins were expressed in COS-1 cells. Immunoelectrophoretic blot analysis was used to compare the levels of expression of the mutant proteins, and their ability to hydrolyze bradykinin and Gly-Pro-hydroxyPro was assessed. Asp449, Asp460, His523, Glu554, and Glu568 are predicted to serve as metal ion ligands in the active site, and mutagenesis of these residues resulted in fully glycosylated proteins that were catalytically inactive. Mutation of His429 and His532 also resulted in catalytically inactive proteins, and these residues, by analogy with E. coli AP-P, are likely to play a role in shuttling protons during catalysis. These studies indicate that mammalian membrane-bound AP-P has an active-site configuration similar to that of other members of the peptidase clan MG, which is compatible with either a dual metal ion model or a single metal ion in the active site. The latter model is consistent, however, with the known metal stoichiometry of both the membrane-bound and cytosolic forms of AP-P and with a recently proposed model for methionyl aminopeptidase.     

Crystal structure determination of cholesterol oxidase from Streptomyces and structural characterization of key active site mutants

Cholesterol oxidase is a monomeric flavoenzyme which catalyzes the oxidation and isomerization of cholesterol to cholest-4-en-3-one. The enzyme interacts with lipid bilayers in order to bind its steroid substrate. The X-ray structure of the enzyme from Brevibacterium sterolicum revealed two loops, comprising residues 78-87 and residues 433-436, which act as a lid over the active site and facilitate binding of the substrate [Vrielink et al. (1991) J. Mol. Biol. 219, 533-554; Li et al. (1993) Biochemistry 32, 11507-11515]. It was postulated that these loops must open, forming a hydrophobic channel between the membrane and the active site of the protein and thus sequestering the cholesterol substrate from the aqueous environment. Here we describe the three-dimensional structure of the homologous enzyme from Streptomyces refined to 1.5 A resolution. Structural comparisons to the enzyme from B. sterolicum reveal significant conformational differences in these loop regions; in particular, a region of the loop comprising residues 78-87 adopts a small amphipathic helical turn with hydrophobic residues directed toward the active site cavity and hydrophilic residues directed toward the external surface of the molecule. It seems reasonable that this increased rigidity reduces the entropy loss that occurs upon binding substrate. Consequently, the Streptomyces enzyme is a more efficient catalyst. In addition, we have determined the structures of three active site mutants which have significantly reduced activity for either the oxidation (His447Asn and His447Gln) or the isomerization (Glu361Gln). Our structural and kinetic data indicate that His447 and Glu361 act as general base catalysts in association with conserved water H2O541 and Asn485. The His447, Glu361, H2O541, and Asn485 hydrogen bond network is conserved among other oxidoreductases. This catalytic tetrad appears to be a structural motif that occurs in flavoenzymes that catalyze the oxidation of unactivated alcohols.     

The 1.3-Angstrom-resolution crystal structure of beta-ketoacyl-acyl carrier protein synthase II from Streptococcus pneumoniae

The beta-ketoacyl-acyl carrier protein synthases are members of the thiolase superfamily and are key regulators of bacterial fatty acid synthesis. As essential components of the bacterial lipid metabolic pathway, they are an attractive target for antibacterial drug discovery. We have determined the 1.3 A resolution crystal structure of the beta-ketoacyl-acyl carrier protein synthase II (FabF) from the pathogenic organism Streptococcus pneumoniae. The protein adopts a duplicated betaalphabetaalphabetaalphabetabeta fold, which is characteristic of the thiolase superfamily. The two-fold pseudosymmetry is broken by the presence of distinct insertions in the two halves of the protein. These insertions have evolved to bind the specific substrates of this particular member of the thiolase superfamily. Docking of the pantetheine moiety of the substrate identifies the loop regions involved in substrate binding and indicates roles for specific, conserved residues in the substrate binding tunnel. The active site triad of this superfamily is present in spFabF as His 303, His 337, and Cys 164. Near the active site is an ion pair, Glu 346 and Lys 332, that is conserved in the condensing enzymes but is unusual in our structure in being stabilized by an Mg(2+) ion which interacts with Glu 346. The active site histidines interact asymmetrically with Lys 332, whose positive charge is closer to His 303, and we propose a specific role for the lysine in polarizing the imidazole ring of this histidine. This asymmetry suggests that the two histidines have unequal roles in catalysis and provides new insights into the catalytic mechanisms of these enzymes.     

Mutational analysis of the zinc- and substrate-binding sites in the CphA metallo-beta-lactamase from Aeromonas hydrophila

The subclass B2 CphA (Carbapenemase hydrolysing Aeromonas) beta-lactamase from Aeromonas hydrophila is a Zn(2+)-containing enzyme that specifically hydrolyses carbapenems. In an effort to evaluate residues potentially involved in metal binding and/or catalysis (His(118), Asp(120), His(196) and His(263)) and in substrate specificity (Val(67), Thr(157), Lys(224) and Lys(226)), site-directed mutants of CphA were generated and characterized. Our results confirm that the first zinc ion is in interaction with Asp(120) and His(263), and thus is located in the 'cysteine' zinc-binding site. His(118) and His(196) residues seem to be interacting with the second zinc ion, as their replacement by alanine residues has a negative effect on the affinity for this second metal ion. Val(67) plays a significant role in the binding of biapenem and benzylpenicillin. The properties of a mutant with a five residue (LFKHV) insertion just after Val(67) also reveals the importance of this region for substrate binding. This latter mutant has a higher affinity for the second zinc ion than wild-type CphA. The T157A mutant exhibits a significantly modified activity spectrum. Analysis of the K224Q and N116H/N220G/K224Q mutants suggests a significant role for Lys(224) in the binding of substrate. Lys(226) is not essential for the binding and hydrolysis of substrates. Thus the present paper helps to elucidate the position of the second zinc ion, which was controversial, and to identify residues important for substrate binding.     

The HELLGH motif of rat liver dipeptidyl peptidase III is involved in zinc coordination and the catalytic activity of the enzyme.

The role of the HELLGH (residues 450-455) motif in the sequence of rat dipeptidyl peptidase III (EC 3.4.14.4) was investigated by replacing Glu451 with an alanine or an aspartic acid residue and by replacing His450 and His455 with a tyrosine residue by site-directed mutagenesis. Mutated cDNAs were expressed three or four times in Escherichia coli, and the resulting proteins were purified to apparent homogeneity. None of the expressed mutated proteins exhibited DPP III activity. The mutants of Glu451 contained 1 mol of zinc per mole of protein, but mutants His450 and His455 did not contain significant amounts of zinc as determined by atomic absorption spectrometry. The Leu453-deleted enzyme (having the zinc aminopeptidase motif HExxH-18-E) had almost the same order of binding affinity (for Arg-Arg-2-naphthylamide) as the wild-type enzyme, but the specificity constant was about 10%. These results provide evidence that the suitable number of amino acids included between Glu451 and His455 is three residues for the enzyme activity and confirm that residues His450, His455, and Glu451 are involved in zinc coordination and catalytic activity.     

Structural and functional characterization of mitochondrial EndoG, a sugar non-specific nuclease which plays an important role during apoptosis

Combining sequence analysis, structure prediction, and site-directed mutagenesis, we have investigated the mechanism of catalysis and substrate binding by the apoptotic mitochondrial nuclease EndoG, which belongs to the large family of DNA/RNA non-specific betabetaalpha-Me-finger nucleases. Catalysis of phosphodiester bond cleavage involves several highly conserved amino acid residues, namely His143, Asn174, and Glu182 required for water activation and metal ion binding, as well as Arg141 required for proper substrate binding and positioning, respectively. These results indicate that EndoG basically follows a similar mechanism as the Serratia nuclease, the best studied representative of the family of DNA/RNA non-specific nucleases, but that differences are observed for transition state stabilisation. In addition, we have identified two putative DNA/RNA binding residues of bovine EndoG, Arg135 and Arg186, strictly conserved only among mammalian members of the nuclease family, suggesting a similar mode of binding to single and double-stranded nucleic acid substrates by these enzymes. Finally, we demonstrate by ectopic expression of active and inactive variants of bovine EndoG in HeLa and CV1-cells that extramitochondrial active EndoG by itself induces cell death, whereas expression of an enzymatically inactive variant does not.     

The active site architecture of Pisum sativum beta-carbonic anhydrase is a mirror image of that of alpha-carbonic anhydrases

We have determined the structure of the beta-carbonic anhydrase from the dicotyledonous plant Pisum sativum at 1.93 A resolution, using a combination of multiple anomalous scattering off the active site zinc ion and non-crystallographic symmetry averaging. The mol- ecule assembles as an octamer with a novel dimer of dimers of dimers arrangement. Two distinct patterns of conservation of active site residues are observed, implying two potentially mechanistically distinct classes of beta-carbonic anhydrases. The active site is located at the interface between two monomers, with Cys160, His220 and Cys223 binding the catalytic zinc ion and residues Asp162 (oriented by Arg164), Gly224, Gln151, Val184, Phe179 and Tyr205 interacting with the substrate analogue, acetic acid. The substrate binding groups have a one to one correspondence with the functional groups in the alpha-carbonic anhydrase active site, with the corresponding residues being closely superimposable by a mirror plane. Therefore, despite differing folds, alpha- and beta-carbonic anhydrase have converged upon a very similar active site design and are likely to share a common mechanism.     

Crystal structure of D-psicose 3-epimerase from Agrobacterium tumefaciens and its complex with true substrate D-fructose: a pivotal role of metal in catalysis, an active site for the non-phosphorylated substrate, and its conformational changes

D-psicose, a rare sugar produced by the enzymatic reaction of D-tagatose 3-epimerase (DTEase), has been used extensively for the bioproduction of various rare carbohydrates. Recently characterized D-psicose 3-epimerase (DPEase) from Agrobacterium tumefaciens was found to belong to the DTEase family and to catalyze the interconversion of D-fructose and D-psicose by epimerizing the C-3 position, with marked efficiency for D-psicose. The crystal structures of DPEase and its complex with the true substrate D-fructose were determined; DPEase is a tetramer and each monomer belongs to a TIM-barrel fold. The active site in each subunit is distinct from that of other TIM-barrel enzymes, which use phosphorylated ligands as the substrate. It contains a metal ion with octahedral coordination to two water molecules and four residues that are absolutely conserved across the DTEase family. Upon binding of D-fructose, the substrate displaces water molecules in the active site, with a conformation mimicking the intermediate cis-enediolate. Subsequently, Trp112 and Pro113 in the beta4-alpha4 loop undergo significant structural changes, sealing off the active site. Structural evidence and site-directed mutagenesis of the putative catalytic residues suggest that the metal ion plays a pivotal role in catalysis by anchoring the bound D-fructose, and Glu150 and Glu244 carry out an epimerization reaction at the C-3 position.     

Direct and indirect roles of His‐418 in metal binding and in the activity of β‐galactosidase (E. coli)

The active site of ß‐galactosidase (E. coli) contains a Mg²⁺ ion ligated by Glu‐416, His‐418 and Glu‐461 plus three water molecules. A Na⁺ ion binds nearby. To better understand the role of the active site Mg²⁺ and its ligands, His‐418 was substituted with Asn, Glu and Phe. The Asn‐418 and Glu‐418 variants could be crystallized and the structures were shown to be very similar to native enzyme. The Glu‐418 variant showed increased mobility of some residues in the active site, which explains why the substitutions at the Mg²⁺ site also reduce Na⁺ binding affinity. The Phe variant had reduced stability, bound Mg²⁺ weakly and could not be crystallized. All three variants have low catalytic activity due to large decreases in the degalactosylation rate. Large decreases in substrate binding affinity were also observed but transition state analogs bound as well or better than to native. The results indicate that His‐418, together with the Mg²⁺, modulate the central role of Glu‐461 in binding and as a general acid/base catalyst in the overall catalytic mechanism. Glucose binding as an acceptor was also dramatically decreased, indicating that His‐418 is very important for the formation of allolactose (the natural inducer of the lac operon).     

The transthyretin-related protein: structural investigation of a novel protein family

The transthyretin-related protein (TRP) family comprises proteins predicted to be structurally related to the homotetrameric transport protein transthyretin (TTR). The function of TRPs is not yet fully established, but recent data suggest that they are involved in purine catabolism. We have determined the three-dimensional structure of the Escherichia coli TRP in two crystal forms; one at 1.65 A resolution in the presence of zinc, and the other at 2.1 A resolution in the presence of zinc and bromide. The structures revealed five zinc-ion-binding sites per monomer. Of these, the zinc ions bound at sites I and II are coordinated in tetrahedral geometries to the side chains of residues His9, His96, His98, Ser114, and three water molecules at the putative ligand-binding site. Of these four residues, His9, His98, and Ser114 are conserved. His9 and His98 bind the central zinc (site I) together with two water molecules. The side chain of His98 also binds to the zinc ion at site II. Bromide ions bind at site I only, replacing one of the water molecules coordinated to the zinc ion. The C-terminal four amino acid sequence motif Y-[RK]-G-[ST] constitutes the signature sequence of the TRP family. Two Tyr111 residues form direct hydrogen bonds to each other over the tetramer interface at the area, which in TTR constitutes the rear part of its thyroxine-binding channel. The putative substrate/ligand-binding channel of TRP is consequently shallower and broader than its counterpart in TTR.     

Complexes of mutants of Escherichia coli aminopeptidase P and the tripeptide substrate ValProLeu

Aminopeptidase P (APPro) is a manganese-containing enzyme that catalyses the hydrolysis of the N-terminal residue of a polypeptide if the second residue is proline. Structures of APPro mutants with reduced or negligible activity have been determined in complex with the tripeptide substrate ValProLeu. In the complex of Glu383Ala APPro with ValProLeu one of the two metal sites is only partly occupied, indicating an essential role for Glu383 in metal binding in the presence of substrate. His361Ala APPro clearly possesses residual activity as the ValProLeu substrate has been cleaved in the crystals; difference electron density consistent with bound ProLeu dipeptide and a disordered Val amino acid is present at the active site. Contrary to previous suggestions, the His243Ala mutant is capable of binding substrate. The structure of the His243Ala APPro complex with ValProLeu shows that the peptide interacts with one of the active-site metal atoms via its terminal amino group. The implications of these complexes for the roles of the respective residues in APPro catalysis are discussed.     

Roles of dimerization domain residues in binding and catalysis by aminoacylase-1

The aminoacylase-1/metallopeptidase 20 (Acy1/M20) family is the largest metallopeptidase family. Several crystal structures feature a metal-binding and a dimerization-mediating domain, both arranged in an extended open conformation. We have recently shown [Lindner et al. (2003) J. Biol. Chem. 278, 44496-44504] that in human Acy1 the invariant residues Glu147 and His206 from the metal-binding and the dimerization domain, respectively, are recruited to the active site from opposite dimer subunits. We hypothesized that, to facilitate this, formation of the binary complex is associated with domain closure, which would also position additional residues in the functional active site of Acy1. These would include two partially conserved dimerization domain residues: an asparagine (Asn263) and an arginine (Arg276) from the same subunit as His206 and Glu147, respectively. In this paper, we investigate the significance of the three dimerization domain residues of human Acy1 His206, Asn263, and Arg276 and, additionally, the nearby Asp274 for catalysis using site-directed mutagenesis. Enzyme complementation assays confirm the putative subunit allocations of these residues, and steady-state kinetics support roles for all of them in catalysis but only involve the Arg276 in substrate-binding. The results are consistent with a model of the closed conformation for the structure of the related enzyme carboxypeptidase G2. This study demonstrates experimentally for the first time for a member of the Acy1/M20 family that several residues outside of the metal-binding domain are involved in binding and catalysis.