Second-site suppression of regulatory phosphorylation in Escherichia coli isocitrate dehydrogenase

Inactivation of Escherichia coli isocitrate dehydrogenase upon phosphorylation at S113 depends upon the direct electrostatic repulsion of the negatively charged gamma-carboxylate of isocitrate by the negatively charged phosphoserine. The effect is mimicked by replacing S113 with aspartate or glutamate, which reduce performance (kcat/K(i).isocitrat/ Km.NADP) by a factor of 10(7). Here, we demonstrate that the inactivating effects of the electrostatic repulsion are completely eliminated by a second-site mutation, and provide the structural basis for this striking example of intragenic suppression. N115 is adjacent to S113 on one face of the D-helix, interacts with isocitrate and NADP+, and has been postulated to serve in both substrate binding and in catalysis. The single N115L substitution reduces affinity for isocitrate by a factor of 50 and performance by a factor of 500. However, the N115L substitution completely suppresses the inactivating electrostatic effects of S113D or S113E: the performance of the double mutants is 10(5) higher than the S113D and S113E single mutants. These mutations have little effect on the kinetics of alternative substrates, which lack the charged gamma-carboxylate of isocitrate. Both glutamate and aspartate at site 113 remain fully ionized in the presence of leucine. In the crystal structure of the N115L mutant, the leucine adopts a different conformer from the wild-type asparagine. Repacking around the leucine forces the amino-terminus of the D-helix away from the rest of the active site. The hydrogen bond between E113 and N115 in the S113E single mutant is broken in the S113E/N115L mutant, allowing the glutamate side chain to move away from the gamma-carboxylate of isocitrate. These movements increase the distance between the carboxylates, diminish the electrostatic repulsion, and lead to the remarkably high activity of the S113E/N115L mutant.     

Access to phosphorylation in isocitrate dehydrogenase may occur by domain shifting

To clarify further the mechanism of regulation by phosphorylation of isocitrate dehydrogenase, cocrystallization of isocitrate dehydrogenase and isocitrate dehydrogenase kinase/phosphatase in the presence of an ATP analog was attempted. Although cocrystallization was unsuccessful, a new crystal form of isocitrate dehydrogenase was obtained which provides insight into the phosphorylation mechanism. The new, orthorhombic crystal form of isocitrate dehydrogenase is related to the previously reported tetragonal form largely by an approximately 16 degrees shift of a large domain relative to the small domain and clasp region within each subunit of the dimeric enzyme. The NADP+ cofactor binding surface is significantly disrupted by the shift to the open conformation. The solvent-accessible surface area and surface-enclosed volume increase by 2% relative to the dimeric tetragonal form. Most of the increase results from expansion of the active site cleft such that the distance across its opening increases from approximately 5 to 13 A, significantly increasing accessibility to Ser-113. The conformation of isocitrate dehydrogenase in the orthorhombic crystal form more closely resembles that of the crystal structure of the homologous enzyme 3-isopropylmalate dehydrogenase than does the tetragonal isocitrate dehydrogenase conformation. Since the crystal lattice forces are fairly weak, it appears that isocitrate dehydrogenase is a flexible molecule that can easily undergo domain shifts and possibly other induced fit conformational changes, to accommodate binding to isocitrate dehydrogenase kinase/phosphatase.     

A theoretical study of torsional flexibility in the active site of aspartic proteinases: implications for catalysis

We have performed ab initio Hartree-Fock self-consistent field calculations on the active site of endothiapepsin. The active site was modeled as a formic acid/formate anion moiety (representing the catalytic aspartates, Asp-32 and -215) and a bound water molecule. Residues Gly-34, Ser-35, Gly-217, and Thr-218, which all form hydrogen bonds to the active site, were modeled using formamide and methanol molecules. The water molecule, which is generally believed to function as the attacking nucleophile in catalysis, was allowed to bind to the active site in four distinct configurations. The geometry of each configuration was optimized using two basis sets (4-31G and 4-31G*). The results indicate that in the native enzyme the nucleophilic water is bound in a catalytically inert configuration. However, by rotating the carboxyl group of Asp-32 by about 90 degrees the water molecule can be reorientated to attack the scissile bond of the substrate. A model of the bound enzyme-substrate complex was constructed from the crystal structure of a difluorostatone inhibitor complexed with endothiapepsin. This model suggests that the substrate itself initiates the reorientation of the nucleophilic water immediately prior to catalysis by forcing the carboxyl group of Asp-32 to rotate. The theoretical results predict that the active site of endothiapepsin undergoes a large distortion during substrate binding and this observation has been used to explain some of the kinetics results which have been reported for mutant aspartic proteinases.     

Mechanism of enolase: the crystal structure of asymmetric dimer enolase-2-phospho-D-glycerate/enolase-phosphoenolpyruvate at 2.0 A resolution

Enolase, a glycolytic enzyme that catalyzes the dehydration of 2-phospho-d-glycerate (PGA) to form phosphoenolpyruvate (PEP), is a homodimer in all eukaryotes and many prokaryotes. Here, we report the crystal structure of a complex between yeast enolase and an equilibrium mixture of PGA and PEP. The structure has been refined using 29 854 reflections with an F/sigma(F) of >/=3 to an R of 0.137 with average deviations of bond lengths and bond angles from ideal values of 0.013 A and 3.1 degrees , respectively. In this structure, the dimer constitutes the crystallographic asymmetric unit. The two subunits are similar, and their superposition gives a rms distance between Calpha atoms of 0.91 A. The exceptions to this are the catalytic loop Val153-Phe169 where the atomic positions in the two subunits differ by up to 4 A and the loop Ser250-Gln277, which follows the catalytic loop Val153-Phe169. In the first subunit, the imidazole side chain of His159 is in contact with the phosphate group of the substrate/product molecule; in the other it is separated by water molecules. A series of hydrogen bonds leading to a neighboring enolase dimer can be identified as being responsible for ordering and stabilization of the conformationally different subunits in the crystal lattice. The electron density present in the active site suggests that in the active site with the direct ligand-His159 hydrogen bond PGA is predominantly bound while in the active site where water molecules separate His159 from the ligand the binding of PEP dominates. The structure indicates that the water molecule hydrating carbon-3 of PEP in the PEP --> PGA reaction is activated by the carboxylates of Glu168 and Glu211. The crystals are unique because they have resolved two intermediates on the opposite sides of the transition state.     

Investigation of the enzymatic mechanism of the yeast chorismate mutase by docking a transition state analog

The structure of the complex of the chorismate mutase from the yeast Saccharomyces cerevisiae with a transition state analog is constructed using a suite of docking tools. The construction finds the best location for the active site in the enzyme, and the best orientation of the analog compound in the active site. The resulting complex shows extensive salt links and hydrogen bonds between the enzyme and the compound, including those mediated by water molecules. A network of polar interactions between amino acid residues is found to solidify the active site of the enzyme. The enzymatic mechanism suggested for a bacterial chorismate mutase, that the active site is by design capable of selecting an active conformer of the substrate, and of stabilizing the transition state, is apparently intact in the yeast enzyme. No direct evidence is found to support an alternative mechanism which involves specific catalytic groups, although the possibility is not eliminated. This finding reinforces the notion of a function being evolutionarily conserved via a common mechanism, rather than via sequential or structural homology.     

Crystal structures of the inactive D30N mutant of feline immunodeficiency virus protease complexed with a substrate and an inhibitor

Crystal structures of complexes of a D30N mutant of feline immunodeficiency virus protease (FIV PR) complexed with a statine-based inhibitor (LP-149), as well as with a substrate based on a modification of this inhibitor (LP-149S), have been solved and refined at resolutions of 2.0 and 1.85 A, respectively. Both the inhibitor and the substrate are bound in the active site of the mutant protease in a similar mode, which also resembles the mode of binding of LP-149 to the native protease. The carbonyl oxygen of the scissile bond in the substrate is not hydrated and is located within the distance of a hydrogen bond to an amido nitrogen atom from one of the two asparagines in the active site of the enzyme. The nitrogen atom of the scissile bond is 3.25 A from the conserved water molecule (Wat301). A model of a tetrahedral intermediate bound to the active site of the native enzyme was built by considering the interactions observed in all three crystal structures of FIV PR. Molecular dynamics simulations of this model bound to native wild-type FIV PR were carried out, to investigate the final stages of the catalytic mechanism of aspartic proteases.     

Crystal structure of carbonic anhydrase from Neisseria gonorrhoeae and its complex with the inhibitor acetazolamide

The crystal structure of carbonic anhydrase from Neisseria gonorrhoeae has been solved to a resolution of 1.78 A by molecular replacement using human carbonic anhydrase II as a template. After refinement the R factor was 17.8% (Rfree=23.2%). There are two molecules per asymmetric unit (space group P21), but they have essentially identical structures. The fold of the N. gonorrhoeae enzyme is very similar to that of human isozyme II; 192 residues, 74 of which are identical in the two enzymes, have equivalent positions in the three-dimensional structures. This corresponds to 85% of the entire polypeptide chain of the bacterial enzyme. The only two cysteine residues in the bacterial enzyme, which has a periplasmic location in the cell, are connected by a disulfide bond. Most of the secondary structure elements present in human isozyme II are retained in N. gonorrhoeae carbonic anhydrase, but there are also differences, particularly in the few helical regions. Long deletions in the bacterial enzyme relative to human isozyme II have resulted in a considerable shortening of three surface loops. One of these deletions, corresponding to residues 128 to 139 in the human enzyme, leads to a widening of the entrance to the hydrophobic part of the active site cavity. Practically all the amino acid residues in the active site of human isozyme II are conserved in the N. gonorrhoeae enzyme and have similar structural positions. However, the imidazole ring of a histidine residue, which has been shown to function as a proton shuttle in the catalytic mechanism of the human enzyme, interacts with an extraneous entity, which has tentatively been identified as a 2-mercaptoethanol molecule from the crystallization medium. When this entity is removed by soaking the crystal in a different medium, the side-chain of His66 becomes quite mobile. The structure of a complex with the sulfonamide inhibitor, acetazolamide, has also been determined. Its position in the active site is very similar to that observed in human carbonic anhydrase II.     

Function and expression of yeast mitochondrial NAD- and NADP-specific isocitrate dehydrogenases

The three isozymes of isocitrate dehydrogenase in Saccharomyces cerevisiae differ in subunit structure, subcellular location, and cofactor specificity. The two mitochondrial isozymes, IDH and IDP1, are NAD- and NADP-specific, respectively. Several lines of evidence presented here confirm the importance of IDH to respiratory processes. Expression of IDH RNA and protein is low with growth on glucose and is elevated with growth on non-fermentable carbon sources, a pattern of expression similar to that seen for other tricarboxylic acid cycle enzymes. In addition, a disruption mutant lacking IDH activity exhibits reduced growth rates on non-fermentable carbon sources, and mitochondria isolated from this mutant are incapable of respiration with added citrate. In contrast, IDP1 expression levels appear to be unresponsive to carbon source, and an IDP1 disruption mutant is not significantly impaired for growth or mitochondrial respiration. These results strongly suggest that IDP1 is incapable of participating in tricarboxylic acid cycle-based respiration despite its mitochondrial location. Analysis of the IDP1 and IDH disruption mutants for glutamate auxotrophy showed that either enzyme can contribute alpha-ketoglutarate for endogenous glutamate synthesis. IDH expression levels were found to be repressed in response to added glutamate during growth on glucose, while IDP1 expression levels remained unchanged. A double mutant lacking both IDP1 and IDH activities proved to be auxotrophic for glutamate during growth on glucose, but was capable of growth independent of added glutamate on non-fermentable carbon sources. These results suggest that the cytosolic NADP-specific IDP2 isozyme may provide alpha-ketoglutarate both for tricarboxylic acid cycle carbon flux and for cytosolic glutamate synthesis during growth on non-fermentable carbon sources in the absence of mitochondrial isocitrate dehydrogenase activity.     

High-resolution crystal structures of delta5-3-ketosteroid isomerase with and without a reaction intermediate analogue

Bacterial Delta5-3-ketosteroid isomerase (KSI) catalyzes a stereospecific isomerization of steroid substrates at an extremely fast rate, overcoming a large disparity of pKa values between a catalytic residue and its target. The crystal structures of KSI from Pseudomonas putida and of the enzyme in complex with equilenin, an analogue of the reaction intermediate, have been determined at 1.9 and 2.5 A resolution, respectively. The structures reveal that the side chains of Tyr14 and Asp99 (a newly identified catalytic residue) form hydrogen bonds directly with the oxyanion of the bound inhibitor in a completely apolar milieu of the active site. No water molecule is found at the active site, and the access of bulk solvent is blocked by a layer of apolar residues. Asp99 is surrounded by six apolar residues, and consequently, its pKa appears to be elevated as high as 9.5 to be consistent with early studies. No interaction was found between the bound inhibitor and the residue 101 (phenylalanine in Pseudomonas testosteroni and methionine in P. putida KSI) which was suggested to contribute significantly to the rate enhancement based on mutational analysis. This observation excludes the residue 101 as a potential catalytic residue and requires that the rate enhancement should be explained solely by Tyr14 and Asp99. Kinetic analyses of Y14F and D99L mutant enzymes demonstrate that Tyr14 contributes much more significantly to the rate enhancement than Asp99. Previous studies and the structural analysis strongly suggest that the low-barrier hydrogen bond of Tyr14 (>7.1 kcal/mol), along with a moderate strength hydrogen bond of Asp99 ( approximately 4 kcal/mol), accounts for the required energy of 11 kcal/mol for the transition-state stabilization.     

Determination of the gene sequence and the three-dimensional structure at 2.4 angstroms resolution of methanol dehydrogenase from Methylophilus W3A1

The DNA sequences for the genes encoding the heavy and light subunits of methanol dehydrogenase from Methylophilus methylotrophus W3A1 have been determined. The deduced amino acid sequence has enabled the structure of the enzyme to be refined at 2.4 angstrom resolution against X-ray data collected on a Hamlin area detector. The structure was refined using the programs PROFFT and X-PLOR with several model building step interspersed. The final model contains two heavy chains (571 amino acids), two light chains (69 amino acids), two molecules of pyrroloquinoline quinone, two Ca2+ and 521 solvent molecules. Each half molecule contains four disulfide linkages and four cis peptides. One of the disulfides is formed from two adjacent cysteine residues linked by a trans peptide which creates a novel eight-membered ring. The heavy subunit is an 8-fold beta-propeller, each "blade" of which is a four-stranded antiparallel twisted beta-sheet. The light chain is an elongated subunit stretching across the surface of the heavy subunit, with residues 1 to 32 containing four beta-turns and residues 33 to 62 forming a helix; however, it neither interacts with the active site, nor the other HL dimer and its functional role is obscure. Around the 8-fold beta-propeller there is a repeating pattern of tryptophan residues located in the outer strand of seven of the eight beta-leaflets, each packed between adjacent leaflets. Each of these tryptophan residues is centered in the beta-strand and participates in the main chain hydrogen bonding of the sheet. Five of the seven tryptophan residues have closely similar interactions with the adjacent beta-leaflet including stacking of the tryptophan indole rings against a peptide plane and formation of a hydrogen bond from NE1 of the indole ring to a main-chain carbonyl. This repeating pattern is conserved over a number of MEDH sequences. The PQQ is located on the pseudo 8-fold rotation axis of the heavy subunit, in a funnel-shaped internal cavity, sandwiched between the indole ring of Trp237 and the two sulfur atoms of the Cys103-Cys104 vicinal disulfide. A hexacoordinate Ca2+ is bound in the active site by one nitrogen and five oxygen ligands, three from the PQQ and the others from two protein side-chains. In the active site an isolated solvent molecule is bound to the O5 of PQQ and to a nearby aspartate side-chain; its position may be the binding site for methanol. The aspartate might than serve as a general base for proton abstraction from the substrate hydroxyl. The C5 atom of PQQ could be activated by electrophilic catalysis by a nearby argenine side-chain or by the calcium ion bound to PQQ.     

Cytosolic isocitrate dehydrogenase in humans, mice, and voles and phylogenetic analysis of the enzyme family

In this study, we report cDNA sequences of the cytosolic NADP-dependent isocitrate dehydrogenase for humans, mice, and two species of voles (Microtus mexicanus and Microtus ochrogaster). Inferred amino acid sequences from these taxa display a high level of amino acid sequence conservation, comparable to that of myosin beta heavy chain, and share known structural features. A Caenorhabditis elegans enzyme that was previously identified as a protein similar to isocitrate dehydrogenase is most likely the NADP-dependent cytosolic isocitrate dehydrogenase enzyme equivalent, based on amino acid similarity to mammalian enzymes and phylogenetic analysis. We also suggest that NADP-dependent isocitrate dehydrogenases characterized from alfalfa, soybean, and eucalyptus are most likely cytosolic enzymes. The phylogenetic tree of various isocitrate dehydrogenases from eukaryotic sources revealed that independent gene duplications may have given rise to the cytosolic and mitochondrial forms of NADP-dependent isocitrate dehydrogenase in animals and fungi. There appears to be no statistical support for a hypothesis that the mitochondrial and cytosolic forms of the enzyme are orthologous in these groups. A possible scenario of the evolution of NADP-dependent isocitrate dehydrogenases is proposed.     

Control of isocitrate lyase synthesis in Chlorella fusca var. vacuolata. The basal activity of the enzyme and the kinetics of induction

1. Isocitrate lyase activity was measured in non-induced Chlorella fusca var. vacuolata cells. 2. During exponential autotrophic growth about 1-2 molecules of the enzyme per cell were present. 3. In light-limited cultures the amount of the enzyme increased to 10-20 molecules/cell. 4. When autotrophic cultures were placed in the dark, the basal activity of isocitrate lyase increased after a 2h lag so that after 8h in the dark there was a 500-fold increase in activity. 5. When isocitrate lyase was induced (by addition of acetate and removal of illumination) in autotrophic cultures which had been growing exponentially, the full induced rate of enzyme synthesis was obtained after 70-80min. 6. When light-limited autotrophic cultures were induced, the rate of isocitrate lyase synthesis was maximal after only 40-50min. 7. These data are consistent with a catabolite-repression control co-ordinated with photosynthetic activity,which may be independent of the specific inducing effect of acetate.     

Non-hydrated state of the acyl phosphate group in the phosphorylated intermediate of (Na+,K+)-ATPase

The position in the acyl phosphate linkage of the phosphorylated intermediate of (Na+, K+)-ATPase that is cleaved by N-methylhydroxylamine was compared with that of the model compound acetylphosphate. The products of the cleavage of the phosphoenzyme by methylhydroxylamine were the active enzyme and a N-P compound, not the inhibited enzyme and inorganic phosphate. This means that the bond cleaved by methylhydroxylamine was the O-P bond, not the C-O bond. In contrast, methylhydroxylamine did not cleave the O-P bond of acetylphosphate in solution, at pH values from 0.3 to 7.0, whether or not the phosphoryl group formed a complex with magnesium. Acetylphosphate and hydroxylamine formed acetohydroxamic acid. Therefore, the state of the acyl phosphate bond in the native phosphoenzyme and in acetylphosphate in solution was different, and the difference was not due to different dissociation states of their phosphoryl groups or the binding of magnesium to the phosphoenzyme. Molecular orbital calculations for acetylphosphate revealed that the phosphorus atom charge is more positive than the carbon atom, irrespective of the dissociation state of the phosphoryl group. Similarly, the overlapping electron population of the O-P bond is always smaller than that of the C-O bond. Thus, the electronic structure of the acyl phosphate linkage of acetylphosphate under vacuum supports the results obtained with the native phosphoenzyme, rather than those obtained with acetylphosphate in solution. The linkage in the active site of the phosphorylated intermediate of (Na+,K+)-ATPase appeared to be equivalent to the non-hydrated state of the model compound acetylphosphate. The phosphoenzyme with bound ouabain, or without a tightly bound divalent cation was insensitive to methylhydroxylamine. The native phosphoenzyme of (Ca2+)-ATPase was not susceptible to methylhydroxylamine.     

Sources of NADPH and expression of mammalian NADP+-specific isocitrate dehydrogenases in Saccharomyces cerevisiae

To compare roles of specific enzymes in supply of NADPH for cellular biosynthesis, collections of yeast mutants were constructed by gene disruptions and matings. These mutants include haploid strains containing all possible combinations of deletions in yeast genes encoding three differentially compartmentalized isozymes of NADP+-specific isocitrate dehydrogenase and in the gene encoding glucose-6-phosphate dehydrogenase (Zwf1p). Growth phenotype analyses of the mutants indicate that either cytosolic NADP+-specific isocitrate dehydrogenase (Idp2p) or the hexose monophosphate shunt is essential for growth with fatty acids as carbon sources and for sporulation of diploid strains, a condition associated with high levels of fatty acid synthesis. No new biosynthetic roles were identified for mitochondrial (Idp1p) or peroxisomal (Idp3p) NADP+-specific isocitrate dehydrogenase isozymes. These and other results suggest that several major presumed sources of biosynthetic reducing equivalents are non-essential in yeast cells grown under many cultivation conditions. To develop an in vivo system for analysis of metabolic function, mammalian mitochondrial and cytosolic isozymes of NADP+-specific isocitrate dehydrogenase were expressed in yeast using promoters from the cognate yeast genes. The mammalian mitochondrial isozyme was found to be imported efficiently into yeast mitochondria when fused to the Idp1p targeting sequence and to substitute functionally for Idp1p for production of alpha-ketoglutarate. The mammalian cytosolic isozyme was found to partition between cytosolic and organellar compartments and to replace functionally Idp2p for production of alpha-ketoglutarate or for growth on fatty acids in a mutant lacking Zwf1p. The mammalian cytosolic isozyme also functionally substitutes for Idp3p allowing growth on petroselinic acid as a carbon source, suggesting partial localization to peroxisomes and provision of NADPH for beta-oxidation of that fatty acid.     

Crystal structure of tyrosine hydroxylase with bound cofactor analogue and iron at 2.3 A resolution: self-hydroxylation of Phe300 and the pterin-binding site

TyrOH is a non-heme iron enzyme which uses molecular oxygen to hydroxylate tyrosine to form L-dihydroxyphenylalanine (L-DOPA), and tetrahydrobiopterin to form 4a-hydroxybiopterin, in the rate-limiting step of the catecholamine biosynthetic pathway. The 2.3 A crystal structure of the catalytic and tetramerization domains of rat tyrosine hydroxylase (TyrOH) in the presence of the cofactor analogue 7,8-dihydrobiopterin and iron shows the mode of pterin binding and the proximity of its hydroxylated 4a carbon to the required iron. The pterin binds on one face of the large active-site cleft, forming an aromatic pi-stacking interaction with Phe300. This phenylalanine residue of TyrOH is found to be hydroxylated in the meta position, most likely through an autocatalytic process, and to consequently form a hydrogen bond to the main-chain carbonyl of Gln310 which anchors Phe300 in the active site. The bound pterin forms hydrogen bonds from N-8 to the main-chain carbonyl of Leu295, from O-4 to Tyr371 and Glu376, from the C-1' OH to the main-chain amides of Leu294 and Leu295, and from the C-2' hydroxyl to an iron-coordinating water. The part of the pterin closest to the iron is the O-4 carbonyl oxygen at a distance of 3.6 A. The iron is 5.6 A from the pterin 4a carbon which is hydroxylated in the enzymatic reaction. No structural changes are observed between the pterin bound and the nonliganded enzyme. On the basis of these structures, molecular oxygen could bind in a bridging position optimally between the pterin C-4a and iron atom prior to substrate hydroxylation. This structure represents the first report of close interactions between pterin and iron in an enzyme active site.     

Structure determination and refinement of human alpha class glutathione transferase A1-1, and a comparison with the Mu and Pi class enzymes

The crystal structure of human alpha class glutathione transferase A1-1 has been determined and refined to a resolution of 2.6 A. There are two copies of the dimeric enzyme in the asymmetric unit. Each monomer is built from two domains. A bound inhibitor, S-benzyl-glutathione, is primarily associated with one of these domains via a network of hydrogen bonds and salt-links. In particular, the sulphur atom of the inhibitor forms a hydrogen bond to the hydroxyl group of Tyr9 and the guanido group of Arg15. The benzyl group of the inhibitor is completely buried in a hydrophobic pocket. The structure shows an overall similarity to the mu and pi class enzymes particularly in the glutathione-binding domain". The main difference concerns the extended C terminus of the alpha class enzyme which forms an extra alpha-helix that blocks one entrance to the active site and makes up part of the substrate binding site.     

Structural characterizations of nonpeptidic thiadiazole inhibitors of matrix metalloproteinases reveal the basis for stromelysin selectivity

The binding of two 5-substituted-1,3,4-thiadiazole-2-thione inhibitors to the matrix metalloproteinase stromelysin (MMP-3) have been characterized by protein crystallography. Both inhibitors coordinate to the catalytic zinc cation via an exocyclic sulfur and lay in an unusual position across the unprimed (P1-P3) side of the proteinase active site. Nitrogen atoms in the thiadiazole moiety make specific hydrogen bond interactions with enzyme structural elements that are conserved across all enzymes in the matrix metalloproteinase class. Strong hydrophobic interactions between the inhibitors and the side chain of tyrosine-155 appear to be responsible for the very high selectivity of these inhibitors for stromelysin. In these enzyme/inhibitor complexes, the S1' enzyme subsite is unoccupied. A conformational rearrangement of the catalytic domain occurs that reveals an inherent flexibility of the substrate binding region leading to speculation about a possible mechanism for modulation of stromelysin activity and selectivity.     

Structural interactions between horseradish peroxidase C and the substrate benzhydroxamic acid determined by X-ray crystallography

The three-dimensional structure of recombinant horseradish peroxidase in complex with BHA (benzhydroxamic acid) is the first structure of a peroxidase-substrate complex demonstrating the existence of an aromatic binding pocket. The crystal structure of the peroxidase-substrate complex has been determined to 2.0 A resolution with a crystallographic R-factor of 0.176 (R-free = 0. 192). A well-defined electron density for BHA is observed in the peroxidase active site, with a hydrophobic pocket surrounding the aromatic ring of the substrate. The hydrophobic pocket is provided by residues H42, F68, G69, A140, P141, and F179 and heme C18, C18-methyl, and C20, with the shortest distance (3.7 A) found between heme C18-methyl and BHA C63. Very little structural rearrangement is seen in the heme crevice in response to substrate binding. F68 moves to form a lid on the hydrophobic pocket, and the distal water molecule moves 0.6 A toward the heme iron. The bound BHA molecule forms an extensive hydrogen bonding network with H42, R38, P139, and the distal water molecule 2.6 A above the heme iron. This remarkably good match in hydrogen bond requirements between the catalytic residues of HRPC and BHA makes the extended interaction between BHA and the distal heme crevice of HRPC possible. Indeed, the ability of BHA to bind to peroxidases, which lack a peripheral hydrophobic pocket, suggests that BHA is a general counterpart for the conserved hydrogen bond donors and acceptors of the distal catalytic site. The closest aromatic residue to BHA is F179, which we predict provides an important hydrophobic interaction with more typical peroxidase substrates.     

Ca2+-dependent structural changes in C-type mannose-binding proteins

C-type animal lectins are a diverse family of proteins which mediate cell-surface carbohydrate-recognition events through a conserved carbohydrate-recognition domain (CRD). Most members of this family possess a carbohydrate-binding activity that depends strictly on the binding of Ca2+ at two sites, designated 1 and 2, in the CRD. The structural transitions associated with Ca2+ binding in C-type lectins have been investigated by determining high-resolution crystal structures of rat serum mannose-binding protein (MBP) bound to one Ho3+ in place of Ca2+, and the apo form of rat liver MBP. The removal of Ca2+ does not affect the core structure of the CRD, but dramatic conformational changes occur in the loops. The most significant structural change in the absence of Ca2+ is the isomerization of a cis-peptide bond preceding a conserved proline residue in Ca2+ site 2. This bond adopts the cis conformation in all Ca2+-bound structures, whereas both cis and trans conformations are observed in the absence of Ca2+. The pattern of structural changes in the three loops that interact with Ca2+ is dictated in large part by the conformation of the prolyl peptide bond. The highly conserved nature of Ca2+ site 2 suggests that the transitions observed in MBPs are general features of Ca2+ binding in C-type lectins.