Kinetic and crystallographic analysis of mutant Escherichia coli aminopeptidase P: insights into substrate recognition and the mechanism of catalysis

Aminopeptidase P (APPro) is a manganese-dependent enzyme that cleaves the N-terminal amino acid from polypeptides where the second residue is proline. APPro shares a similar fold, substrate specificity, and catalytic mechanism with methionine aminopeptidase and prolidase. To investigate the roles of conserved residues at the active site, seven mutant forms of APPro were characterized kinetically and structurally. Mutation of individual metal ligands selectively abolished binding of either or both Mn(II) atoms at the active site, and none of these metal-ligand mutants had detectable catalytic activity. Mutation of the conserved active site residues His243 and His361 revealed that both are required for catalysis. We propose that His243 stabilizes substrate binding through an interaction with the carbonyl oxygen of the requisite proline residue of a substrate and that His361 stabilizes substrate binding and the gem-diol catalytic intermediate. Sequence, structural, and kinetic analyses reveal that His350, conserved in APPro and prolidase but not in methionine aminopeptidase, forms part of a hydrophobic binding pocket that gives APPro its proline specificity. Further, peptides in which the required proline residue is replaced by N-methylalanine or alanine are cleaved by APPro, but they are extremely poor substrates due to a loss of interactions between the prolidyl ring of the substrate and the hydrophobic proline-binding pocket.     

Studies of specificity and catalysis in trypsin by structural analysis of site-directed mutants

We are probing the determinants of catalytic function and substrate specificity in serine proteases by kinetic and crystallographic characterization of genetically engineered site-directed mutants of rat trypsin. The role of the aspartyl residue at position 102, common to all members of the serine protease family, has been tested by substitution with asparagine. In the native enzyme, Asp102 accepts a hydrogen bond from the catalytic base His57, which facilitates the transfer of a proton from the enzyme nucleophile Ser195 to the substrate leaving group. At neutral pH, the mutant is four orders of magnitude less active than the naturally occurring enzyme, but its binding affinity for model substrates is virtually undiminished. Crystallographic analysis reveals that Asn102 donates a hydrogen bond to His57, forcing it to act as donor to Ser195. Below pH 6, His57 becomes statistically disordered. Presumably, the di-protonated population of histidyl side chains are unable to hydrogen bond to Asn102. Steric conflict may cause His57 to rotate away from the catalytic site. These results suggest that Asp102 not only provides inductive and orientation effects, but also stabilizes the productive tautomer of His57. Three experiments were carried out to alter the substrate specificity of trypsin. Glycine residues at positions 216 and 226 in the substrate-binding cavity were replaced by alanine residues in order to differentially affect lysine and arginine substrate binding. While the rate of catalysis by the mutant enzymes was reduced in the mutant enzymes, their substrate specificity was enhanced relative to trypsin. The increased specificity was caused by differential effects on the catalytic activity towards arginine and lysine substrates. The Gly----Ala substitution at 226 resulted in an altered conformation of the enzyme which is converted to an active trypsin-like conformation upon binding of a substrate analog. In a third experiment, Lys189, at the bottom of the specificity pocket, was replaced with an aspartate with the expectation that specificity of the enzyme might shift to aspartate. The mutant enzyme is not capable of cleaving at Arg and Lys or Asp, but shows an enhanced chymotrypsin-like specificity. Structural investigations of these mutants are in progress.     

X-ray crystal structures of active site mutants of the vanadium-containing chloroperoxidase from the fungus Curvularia inaequalis

The X-ray structures of the chloroperoxidase from Curvularia inaequalis, heterologously expressed in Saccharomyces cerevisiae, have been determined both in its apo and in its holo forms at 1.66 and 2.11?Å resolution, respectively. The crystal structures reveal that the overall structure of this enzyme remains nearly unaltered, particularly at the metal binding site. At the active site of the apo-chloroperoxidase structure a clearly defined sulfate ion was found, partially stabilised through electrostatic interactions and hydrogen bonds with positively charged residues involved in the interactions with the vanadate in the native protein. The vanadate binding pocket seems to form a very rigid frame stabilising oxyanion binding. The rigidity of this active site matrix is the result of a large number of hydrogen bonding interactions involving side chains and the main chain of residues lining the active site. The structures of single site mutants to alanine of the catalytic residue His404 and the vanadium protein ligand His496 have also been analysed. Additionally we determined the structural effects of mutations to alanine of residue Arg360, directly involved in the compensation of the negative charge of the vanadate group, and of residue Asp292 involved in forming a salt bridge with Arg490 which also interacts with the vanadate. The enzymatic chlorinating activity is drastically reduced to approximately 1% in mutants D292A, H404A and H496A. The structures of the mutants confirm the view of the active site of this chloroperoxidase as a rigid matrix providing an oxyanion binding site. No large changes are observed at the active site for any of the analysed mutants. The empty space left by replacement of large side chains by alanines is usually occupied by a new solvent molecule which partially replaces the hydrogen bonding interactions to the vanadate. The new solvent molecules additionally replace part of the interactions the mutated side chains were making to other residues lining the active site frame. When this is not possible, another side chain in the proximity of the mutated residue moves in order to satisfy the hydrogen bonding potential of the residues located at the active site frame.     

Active site specificity of plasmepsin II.

Members of the aspartic proteinase family of enzymes have very similar three-dimensional structures and catalytic mechanisms. Each, however, has unique substrate specificity. These distinctions arise from variations in amino acid residues that line the active site subsites and interact with the side chains of the amino acids of the peptides that bind to the active site. To understand the unique binding preferences of plasmepsin II, an enzyme of the aspartic proteinase class from the malaria parasite, Plasmodium falciparum, chromogenic octapeptides having systematic substitutions at various positions in the sequence were analyzed. This enabled the design of new, improved substrates for this enzyme (Lys-Pro-Ile-Leu-Phe*Nph-Ala/Glu-Leu-Lys, where * indicates the cleavage point). Additionally, the crystal structure of plasmepsin II was analyzed to explain the binding characteristics. Specific amino acids (Met13, Ser77, and Ile287) that were suspected of contributing to active site binding and specificity were chosen for site-directed mutagenesis experiments. The Met13Glu and Ile287Glu single mutants and the Met13Glu/Ile287Glu double mutant gain the ability to cleave substrates containing Lys residues.     

The binding and release of oxygen and hydrogen peroxide are directed by a hydrophobic tunnel in cholesterol oxidase

The usage by enzymes of specific binding pathways for gaseous substrates or products is debated. The crystal structure of the redox enzyme cholesterol oxidase, determined at sub-angstrom resolution, revealed a hydrophobic tunnel that may serve as a binding pathway for oxygen and hydrogen peroxide. This tunnel is formed by a cascade of conformational rearrangements and connects the active site with the exterior surface of the protein. To elucidate the relationship between this tunnel and gas binding and release, three mutant enzymes were constructed to block the tunnel or its putative gate. Mutation of the proposed gating residue Asn485 to Asp or tunnel residue Phe359 or Gly347 to Trp or Asn reduces the catalytic efficiency of oxidation. The K mO 2 increases from 300 +/- 35 microM for the wild-type enzyme to 617 +/- 15 microM for the F359W mutant. The k cat for the F359W mutant-catalyzed reaction decreases 13-fold relative to that of the wild-type-catalyzed reaction. The N485D and G347N mutants could not be saturated with oxygen. Transfer of hydride from the sterol to the flavin prosthetic group is no longer rate-limiting for these tunnel mutants. The steady-state kinetics of both wild-type and tunnel mutant enzymes are consistent with formation of a ternary complex of steroid and oxygen during catalysis. Furthermore, kinetic cooperativity with respect to molecular oxygen is observed with the tunnel mutants, but not with the wild-type enzyme. A rate-limiting conformational change for binding and release of oxygen and hydrogen peroxide, respectively, is consistent with the cooperative kinetics. In the atomic-resolution structure of F359W, the indole ring of the tryptophan completely fills the tunnel and is observed in only a single conformation. The size of the indole is proposed to limit conformational rearrangement of residue 359 that leads to tunnel opening in the wild-type enzyme. Overall, these results substantiate the functional importance of the tunnel for substrate binding and product release.     

The crystal structure of the formiminotransferase domain of formiminotransferase-cyclodeaminase: implications for substrate channeling in a bifunctional enzyme

BACKGROUND: The bifunctional enzyme formiminotransferase-cyclodeaminase (FTCD) contains two active sites at different positions on the protein structure. The enzyme binds a gamma-linked polyglutamylated form of the tetrahydrofolate substrate and channels the product of the transferase reaction from the transferase active site to the cyclodeaminase active site. Structural studies of this bifunctional enzyme and its monofunctional domains will provide insight into the mechanism of substrate channeling and the two catalytic reactions. RESULTS: The crystal structure of the formiminotransferase (FT) domain of FTCD has been determined in the presence of a product analog, folinic acid. The overall structure shows that the FT domain comprises two subdomains that adopt a novel alpha/beta fold. Inspection of the folinic acid binding site reveals an electrostatic tunnel traversing the width of the molecule. The distribution of charged residues in the tunnel provides insight into the possible mode of substrate binding and channeling. The electron density reveals that the non-natural stereoisomer, (6R)-folinic acid, binds to the protein; this observation suggests a mechanism for product release. In addition, a single molecule of glycerol is bound to the enzyme and indicates a putative binding site for formiminoglutamate. CONCLUSIONS: The structure of the FT domain in the presence of folinic acid reveals a possible novel mechanism for substrate channeling. The position of the folinic acid and a bound glycerol molecule near to the sidechain of His82 suggests that this residue may act as the catalytic base required for the formiminotransferase mechanism.     

Selective alteration of substrate specificity by replacement of aspartic acid-189 with lysine in the binding pocket of trypsin

To test the role of Asp-189 which is located at the base of the substrate binding pocket in determining the specificity of trypsin toward basic substrates, this residue was replaced with a lysine residue by site-directed mutagenesis. Both rat trypsinogen and Lys-189 trypsinogen were expressed and secreted into the periplasmic space of Escherichia coli. The proteins were purified to homogeneity and activated by porcine enterokinase, and their catalytic activities were determined on natural and synthetic substrates. Lys-189 trypsin displayed no catalytic activity toward arginyl and lysyl substrates. Further, there was no compensatory change in specificity toward acidic substrates; no cleavage of aspartyl or glutamyl bonds was detected. Additional studies of substrate specificity involving gas-phase sequence analyses of digested natural substrates revealed an inherent but low chymotrypsin-like activity of trypsin. This activity was retained but modified by the Asp to Lys change at position 189. In addition to hydrolyzing phenylalanyl and tyrosyl peptide bonds, the mutant enzyme has the unique property of cleaving leucyl bonds. On the basis of computer graphic modeling studies of the Lys-189 side chain, it appears that the positively charged NH2 group is directed outside the substrate binding pocket. The resulting hydrophobic cavity may explain the altered substrate specificity of the mutant enzyme. The relatively low chymotrypsin-like activity of both recombinant enzymes may be due to distorted positioning of the scissile bond with respect to the catalytic triad rather than to the lack of sufficient interaction between the hydrophobic side chains and the substrate binding pocket of the enzyme.     

The X-ray structure of N-methyltryptophan oxidase reveals the structural determinants of substrate specificity

The X-ray structure of monomeric N-methyltryptophan oxidase from Escherichia coli (MTOX) has been solved at 3.2 A resolution by molecular replacement methods using Bacillus sp. sarcosine oxidase structure (MSOX, 43% sequence identity) as search model. The analysis of the substrate binding site highlights the structural determinants that favour the accommodation of the bulky N-methyltryptophan residue in MTOX. In fact, although the nature and geometry of the catalytic residues within the first contact shell of the FAD moiety appear to be virtually superposable in MTOX and MSOX, the presence of a Thr residue in position 239 in MTOX (Met245 in MSOX) located at the entrance of the active site appears to play a key role for the recognition of the amino acid substrate side chain. Accordingly, a 15 fold increase in k(cat) and 100 fold decrease in K(m) for sarcosine as substrate has been achieved in MTOX upon T239M mutation, with a concomitant three-fold decrease in activity towards N-methyltryptophan. These data provide clear evidence for the presence of a catalytic core, common to the members of the methylaminoacid oxidase subfamily, and of a side chain recognition pocket, located at the entrance of the active site, that can be adjusted to host diverse aminoacids in the different enzyme species. The site involved in the covalent attachment of flavin has also been addressed by screening degenerate mutants in the relevant positions around Cys308-FAD linkage. Lys341 appears to be the key residue involved in flavin incorporation and covalent linkage.     

The x-ray structure of epoxide hydrolase from Agrobacterium radiobacter AD1. An enzyme to detoxify harmful epoxides.

Epoxide hydrolases catalyze the cofactor-independent hydrolysis of reactive and toxic epoxides. They play an essential role in the detoxification of various xenobiotics in higher organisms and in the bacterial degradation of several environmental pollutants. The first x-ray structure of one of these, from Agrobacterium radiobacter AD1, has been determined by isomorphous replacement at 2.1-A resolution. The enzyme shows a two-domain structure with the core having the alpha/beta hydrolase-fold topology. The catalytic residues, Asp107 and His275, are located in a predominantly hydrophobic environment between the two domains. A tunnel connects the back of the active-site cavity with the surface of the enzyme and provides access to the active site for the catalytic water molecule, which in the crystal structure, has been found at hydrogen bond distance to His275. Because of a crystallographic contact, the active site has become accessible for the Gln134 side chain, which occupies a position mimicking a bound substrate. The structure suggests Tyr152/Tyr215 as the residues involved in substrate binding, stabilization of the transition state, and possibly protonation of the epoxide oxygen.     

Structure of the Alkalohyperthermophilic Archaeoglobus fulgidus Lipase Contains a Unique C-Terminal Domain Essential for Long-Chain Substrate Binding

Several crystal structures of AFL, a novel lipase from the archaeon Archaeoglobus fulgidus, complexed with various ligands, have been determined at about 1.8 Å resolution. This enzyme has optimal activity in the temperature range of 70-90 °C and pH 10-11. AFL consists of an N-terminal α/β-hydrolase fold domain, a small lid domain, and a C-terminal β-barrel domain. The N-terminal catalytic domain consists of a 6-stranded β-sheet flanked by seven α-helices, four on one side and three on the other side. The C-terminal lipid binding domain consists of a β-sheet of 14 strands and a substrate covering motif on top of the highly hydrophobic substrate binding site. The catalytic triad residues (Ser136, Asp163, and His210) and the residues forming the oxyanion hole (Leu31 and Met137) are in positions similar to those of other lipases. Long-chain lipid is located across the two domains in the AFL-substrate complex. Structural comparison of the catalytic domain of AFL with a homologous lipase from Bacillus subtilis reveals an opposite substrate binding orientation in the two enzymes. AFL has a higher preference toward long-chain substrates whose binding site is provided by a hydrophobic tunnel in the C-terminal domain. The unusually large interacting surface area between the two domains may contribute to thermostability of the enzyme. Two amino acids, Asp61 and Lys101, are identified as hinge residues regulating movement of the lid domain. The hydrogen-bonding pattern associated with these two residues is pH dependent, which may account for the optimal enzyme activity at high pH. Further engineering of this novel lipase with high temperature and alkaline stability will find its use in industrial applications.     

Further characterization of the putative human isopeptidase T catalytic site

The human isopeptidase T (isoT) is a zinc-binding deubiquitinating enzyme involved in the disassembly of free K48-linked polyubiquitin chains into ubiquitin monomers. The catalytic site of this enzyme is thought to be composed of Cys335, Asp435, His786 and His795. These four residues were site-directed mutagenized. None of the mutants were able to cleave a peptide-linked ubiquitin dimer. Similarly, C335S, D435N and H795N mutants had virtually no activity against a K48-linked isopeptide ubiquitin dimer, which is an isoT-specific substrate that mimics the K48-linked polyubiquitin chains. On the other hand, the H786N mutant retained a partial activity toward the K48-linked substrate, suggesting that the His786 residue might not be part of the catalytic site. None of the mutations significantly affected the capacity of isoT to bind ubiquitin and zinc. Thus, the catalytic site of UBPs could resemble that of other cysteine proteases, which contain one Cys, one Asp and one His.     

Multiple roles of glutathione binding-site residues of glutathione s-transferase

This study was designed to characterize residues in the glutathione binding site of AdGSTD4-4 from the mosquito malaria vector Anopheles dirus. The data revealed that Leu33, His38 and His50 each play a role in enzyme catalysis and glutathione binding. The mutants of these three residues also displayed differences in hydrophobic substrate specificity, suggesting that changes in the active site conformation occurred. Differences in conformations was also suggested by protein stability changes. These results indicate that residues in the glutathione binding site are not only important in the catalytic function but also play a role in the structural integrity of the enzyme.     

Structure and mechanism of 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase. An enzyme in the mevalonate-independent isoprenoid biosynthetic pathway.

The enzyme 2-C-methyl-D-erythritol 2,4-cyclodiphosphate (MECDP) synthase catalyzes the conversion of 4-diphosphocytidyl-2-C-methyl-D-erythritol 2-phosphate (CDP-ME2P) to MECDP, a highly unusual cyclodiphosphate-containing intermediate on the mevalonate-independent pathway to isopentenyl diphosphate and dimethylallyl diphosphate. We now report two x-ray crystal structures of MECDP synthase refined to 2.8-A resolution. The first structure contains a bound Mn(2+) cation, and the second structure contains CMP, MECDP, and Mn(2+). The protein adopts a homotrimeric quaternary structure built around a central hydrophobic cavity and three externally facing active sites. Each of these active sites is located between two adjacent monomers. A tetrahedrally arranged transition metal binding site, potentially occupied by Mn(2+), sits at the base of the active site cleft. A phosphate oxygen of MECDP and the side chains of Asp(8), His(10), and His(42) occupy the metal ion coordination sphere. These structures reveal for the first time the structural determinants underlying substrate, product, and Mn(2+) recognition and the likely catalytic mechanism accompanying the biosynthesis of the cyclodiphosphate-containing isoprenoid precursor, MECDP.     

Hyperactive mutants of mouse d-aspartate oxidase: mutagenesis of the active site residue serine 308

The role of Ser-308 of murine D-aspartate oxidase (mDASPO), particularly its side chain hydroxyl group, was investigated through the use of site-specific mutational analysis of Ser-308. Recombinant mDASPO carrying a substitution of Gly, Ala, or Tyr for Ser-308 was generated, and fused to either His (His-mDASPO), or glutathione S-transferase, His, and S (GHS-mDASPO) at its N-terminus. Wild-type His-mDASPO or GHS-mDASPO or their mutant derivatives were expressed in Escherichia coli and purified by affinity chromatography. All purified recombinant proteins had functional DASPO activity. The Gly-308 and Ala-308 mutants had significantly higher catalytic efficiency towards D-Asp and N-methyl-D-Asp, and a higher affinity for flavin adenine dinucleotide (FAD) compared to the wild-type enzyme. The Tyr-308 mutant had lower catalytic efficiency and binding capacity. These results suggest that the side chain hydroxyl group of a critical residue of mDASPO, Ser-308, down-regulates enzymatic activity, substrate binding, and FAD binding. This study provides information on the active site of DASPO that will considerably enhance our understanding of the biological significance of this enzyme.     

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.     

Insights into the mechanism of dihydropyrimidine dehydrogenase from site-directed mutagenesis targeting the active site loop and redox cofactor coordination

In mammals, the pyrimidines uracil and thymine are metabolised by a three-step reductive degradation pathway. Dihydropyrimidine dehydrogenase (DPD) catalyses its first and rate-limiting step, reducing uracil and thymine to the corresponding 5,6-dihydropyrimidines in an NADPH-dependent reaction. The enzyme is an adjunct target in cancer therapy since it rapidly breaks down the anti-cancer drug 5-fluorouracil and related compounds. Five residues located in functionally important regions were targeted in mutational studies to investigate their role in the catalytic mechanism of dihydropyrimidine dehydrogenase from pig. Pyrimidine binding to this enzyme is accompanied by active site loop closure that positions a catalytically crucial cysteine (C671) residue. Kinetic characterization of corresponding enzyme mutants revealed that the deprotonation of the loop residue H673 is required for active site closure, while S670 is important for substrate recognition. Investigations on selected residues involved in binding of the redox cofactors revealed that the first FeS cluster, with unusual coordination, cannot be reduced and displays no activity when Q156 is mutated to glutamate, and that R235 is crucial for FAD binding.     

Key substrate recognition residues in the active site of a plant cytochrome P450, CYP73A1. Homology guided site-directed mutagenesis.

CYP73 enzymes are highly conserved cytochromes P450 in plant species that catalyse the regiospecific 4-hydroxylation of cinnamic acid to form precursors of lignin and many other phenolic compounds. A CYP73A1 homology model based on P450 experimentally solved structures was used to identify active site residues likely to govern substrate binding and regio-specific catalysis. The functional significance of these residues was assessed using site-directed mutagenesis. Active site modelling predicted that N302 and I371 form a hydrogen bond and hydrophobic contacts with the anionic site or aromatic ring of the substrate. Modification of these residues led to a drastic decrease in substrate binding and metabolism without major perturbation of protein structure. Changes to residue K484, which is located too far in the active site model to form a direct contact with cinnamic acid in the oxidized enzyme, did not influence initial substrate binding. However, the K484M substitution led to a 50% loss in catalytic activity. K484 may affect positioning of the substrate in the reduced enzyme during the catalytic cycle, or product release. Catalytic analysis of the mutants with structural analogues of cinnamic acid, in particular indole-2-carboxylic acid that can be hydroxylated with different regioselectivities, supports the involvement of N302, I371 and K484 in substrate docking and orientation.     

Three-dimensional structure of horse liver alcohol dehydrogenase at 2-4 A resolution.

The crystal structure analysis of horse liver alcohol dehydrogenase has been extended to 2.4 Å resolution. From the corresponding electron density map of the apoenzyme we have determined the positions of the 374 amino acids in the polypeptide chain of each subunit.The coenzyme binding domain of the subunit comprises residues 176 to 318. 45% of these residues are helical and 32% are in the central six-stranded pleated sheet structure. The positions and orientations of the helices with respect to the pleated sheet indicate a possible folding mechanism for this part of the subunit structure. The coenzyme analogue ADP-ribose binds to this domain in a position and orientation very similar to coenzyme binding to lactate dehydrogenase. The adenine part binds in a hydrophobic pocket, the adenosine ribose is hydrogen-bonded to the side chain of Asp223, the pyrophosphate is positioned by interaction with Arg47 and the nicotinamide ribose is 6Å away from the catalytic zinc atom.The catalytic domain is mainly built up from three distinct antiparallel pleated-sheet regions. Residues within this domain provide ligands to the catalytic zinc atom; Cys46, His67 and Cys174. An approximate tetrahedral coordination of this zinc is completed by a water molecule or hydroxyl ion depending on the pH. Residues 95 to 113 form a lobe that binds the second zinc atom of the subunit. This zinc is liganded in a distorted tetrahedral arrangement by four sulphur atoms from the cysteine residues 97, 100, 103 and 111. The lobe forms one side of a significant cleft in the enzyme surface suggesting that this region might constitute a second catalytic centre of unknown function.The two domains of the subunit are separated by a crevice that contains a wide and deep hydrophobic pocket. The catalytic zinc atom is at the bottom of this pocket, with the zinc-bound water molecule projecting out into the pocket. This water molecule is hydrogen-bonded to the side chain of Ser48 which in turn is hydrogen-bonded to His51. The pocket which in all probability is the binding site for the substrate and the nicotinamide moiety of the coenzyme, is lined almost exclusively with hydrophobic side chains. Both subunits contribute residues to each of the two substrate binding pockets of the molecule. The only accessible polar groups in the vicinity of the catalytic centre are Ser48 and Thr178 apart from zinc and the zinc-bound water molecule.     

A phosphate-binding subsite in bovine pancreatic ribonuclease A can be converted into a very efficient catalytic site

A general acid-base catalytic mechanism is responsible for the cleavage of the phosphodiester bonds of the RNA by ribonuclease A (RNase A). The main active site is formed by the amino acid residues His12, His119, and Lys41, and the process follows an endonucleolytic pattern that depends on the existence of a noncatalytic phosphate-binding subsite adjacent, on the 3'-side, to the active site; in this region the phosphate group of the substrate establishes electrostatic interactions through the side chains of Lys7 and Arg10. We have obtained, by means of site-directed mutagenesis, RNase A variants with His residues both at positions 7 and 10. These mutations have been introduced with the aim of transforming a noncatalytic binding subsite into a putative new catalytic active site. The RNase activity of these variants was determined by the zymogram technique and steady-state kinetic parameters were obtained by spectrophotometric methods. The variants showed a catalytic efficiency in the same order of magnitude as the wild-type enzyme. However, we have demonstrated in these variants important effects on the substrate's cleavage pattern. The quadruple mutant K7H/R10H/H12K/H119Q shows a clear increase of the exonucleolytic activity; in this case the original native active site has been suppressed, and, as consequence, its activity can only be associated to the new active site. In addition, the mutant K7H/R10H, with two putative active sites, also shows an increase in the exonucleolytic preference with respect to the wild type, a fact that may be correlated with the contribution of the new active site.     

Probing the active center gorge of acetylcholinesterase by fluorophores linked to substituted cysteines

To examine the influence of individual side chains in governing rates of ligand entry into the active center gorge of acetylcholinesterase and to characterize the dynamics and immediate environment of these residues, we have conjugated reactive groups with selected charge and fluorescence characteristics to cysteines substituted by mutagenesis at specific positions on the enzyme. Insertion of side chains larger than in the native tyrosine at position 124 near the constriction point of the active site gorge confers steric hindrance to affect maximum catalytic throughput (k(cat)/K(m)) and rates of diffusional entry of trifluoroketones to the active center. Smaller groups appear not to present steric constraints to entry; however, cationic side chains selectively and markedly reduce cation ligand entry through electrostatic repulsion in the gorge. The influence of side chain modification on ligand kinetics has been correlated with spectroscopic characteristics of fluorescent side chains and their capacity to influence the binding of a peptide, fasciculin, which inhibits catalysis peripherally by sealing the mouth of the gorge. Acrylodan conjugated to cysteine was substituted for tyrosine at position 124 within the gorge, for histidine 287 on the surface adjacent to the gorge and for alanine 262 on a mobile loop distal to the gorge. The 124 position reveals the most hydrophobic environment and the largest hypsochromic shift of the emission maximum with fasciculin binding. This finding likely reflects a sandwiching of the acrylodan in the complex with the tip of fasciculin loop II. An intermediate spectral shift is found for the 287 position, consistent with partial occlusion by loops II and III of fasciculin in the complex. Spectroscopic properties of the acrylodan at the 262 position are unaltered by fasciculin addition. Hence, combined spectroscopic and kinetic analyses reveal distinguishing characteristics in various regions of acetylcholinesterase that influence ligand association.