Composite active site of chondroitin lyase ABC accepting both epimers of uronic acid.

Enzymes have evolved as catalysts with high degrees of stereospecificity. When both enantiomers are biologically important, enzymes with two different folds usually catalyze reactions with the individual enantiomers. In rare cases a single enzyme can process both enantiomers efficiently, but no molecular basis for such catalysis has been established. The family of bacterial chondroitin lyases ABC comprises such enzymes. They can degrade both chondroitin sulfate (CS) and dermatan sulfate (DS) glycosaminoglycans at the nonreducing end of either glucuronic acid (CS) or its epimer iduronic acid (DS) by a beta-elimination mechanism, which commences with the removal of the C-5 proton from the uronic acid. Two other structural folds evolved to perform these reactions in an epimer-specific fashion: (alpha/alpha)(5) for CS (chondroitin lyases AC) and beta-helix for DS (chondroitin lyases B); their catalytic mechanisms have been established at the molecular level. The structure of chondroitinase ABC from Proteus vulgaris showed surprising similarity to chondroitinase AC, including the presence of a Tyr-His-Glu-Arg catalytic tetrad, which provided a possible mechanism for CS degradation but not for DS degradation. We determined the structure of a distantly related Bacteroides thetaiotaomicron chondroitinase ABC to identify additional structurally conserved residues potentially involved in catalysis. We found a conserved cluster located approximately 12 A from the catalytic tetrad. We demonstrate that a histidine in this cluster is essential for catalysis of DS but not CS. The enzyme utilizes a single substrate-binding site while having two partially overlapping active sites catalyzing the respective reactions. The spatial separation of the two sets of residues suggests a substrate-induced conformational change that brings all catalytically essential residues close together.

Structure of Ca(2+)-loaded human grancalcin.

Grancalcin is a cytosolic Ca(2+)-binding protein originally identified in human neutrophils. It belongs to a new class of EF-hand proteins, called PEF proteins, which contain five EF-hand motifs. At the N-terminus of grancalcin there is a approximately 50 residue-long segment rich in glycines and prolines. The fifth EF-hand, unpaired within the monomer, provides a means for dimerization through pairing with its counterpart in a second molecule. The structure of full-length grancalcin in the apo form and with one EF3 within the dimer occupied by a Ca(2+) ion have been determined. Although the N-terminal segment was present in the molecule, this part was disordered in the crystals. Here, the structure of a truncated form of grancalcin, which is lacking 52 N-terminal residues, in the presence and absence of Ca(2+) is presented. In the Ca(2+)-bound form the ions are found in the EF1 and EF3 hands. Binding of Ca(2+) to these two EF hands produces only minor conformational changes, mostly within the EF1 Ca(2+)-binding loop. This observation supports the hypothesis, formulated on the basis of the structure of a homologous protein ALG-2 which shows significant differences in the orientation of EF4 and EF5 compared with grancalcin, that calcium is a necessary factor but not sufficient alone for inducing a significant conformational change in PEF proteins.

The Structure of calnexin, an ER chaperone involved in quality control of protein folding.

The three-dimensional structure of the lumenal domain of the lectin-like chaperone calnexin determined to 2.9 A resolution reveals an extended 140 A arm inserted into a beta sandwich structure characteristic of legume lectins. The arm is composed of tandem repeats of two proline-rich sequence motifs which interact with one another in a head-to-tail fashion. Identification of the ligand binding site establishes calnexin as a monovalent lectin, providing insight into the mechanism by which the calnexin family of chaperones interacts with monoglucosylated glycoproteins.

Structure of apoptosis-linked protein ALG-2: insights into Ca2+-induced changes in penta-EF-hand proteins.

BACKGROUND: The Ca2+ binding apoptosis-linked gene-2 (ALG-2) protein acts as a proapoptotic factor in a variety of cell lines and is required either downstream or independently of caspases for apoptosis to occur. ALG-2 belongs to the penta-EF-hand (PEF) protein family and has two high-affinity and one low-affinity Ca2+ binding sites. Like other PEF proteins, its N terminus contains a Gly/Pro-rich segment. Ca2+ binding is required for the interaction with the target protein, ALG-2 interacting protein 1 (AIP1). RESULTS: We present the 2.3 A resolution crystal structure of Ca2+-Ioaded des1-20ALG-2 (aa 21-191), which was obtained by limited proteolysis of recombinant ALG-2 with elastase. The molecule contains eight alpha helices that fold into five EF-hands, and, similar to other members of this protein family, the molecule forms dimers. Ca2+ ions bind to EF1, EF3, and, surprisingly, to EF5. In the related proteins calpain and grancalcin, the EF5 does not bind Ca2+ and is thought to primarily facilitate dimerization. Most importantly, the conformation of des1-20ALG-2 is significantly different from that of calpain and grancalcin. This difference can be described as a rigid body rotation of EF1-2 relative to EF4-5 and the dimer interface, with a hinge within the EF3 loop. An electron density, which is interpreted as a hydrophobic Gly/Pro-rich decapeptide that is possibly derived from the cleaved N terminus, was found in a hydrophobic cleft between these two halves of the molecule. CONCLUSIONS: A different relative orientation of the N- and C-terminal halves of des1-20ALG-2 in the presence of Ca2+ and the peptide as compared to other Ca2+loaded PEF proteins changes substantially the shape of the molecule, exposing a hydrophobic patch on the surface for peptide binding and a large cleft near the dimer interface. We postulate that the binding of a Gly/ Pro-rich peptide in the presence of Ca2+ induces a conformational rearrangement in ALG-2, and that this mechanism is common to other PEF proteins.

Crystal structure of histidinol phosphate aminotransferase (HisC) from Escherichia coli, and its covalent complex with pyridoxal-5'-phosphate and l-histidinol phosphate.

The biosynthesis of histidine is a central metabolic process in organisms ranging from bacteria to yeast and plants. The seventh step in the synthesis of histidine within eubacteria is carried out by a pyridoxal-5'-phosphate (PLP)-dependent l-histidinol phosphate aminotransferase (HisC, EC 2.6.1.9). Here, we report the crystal structure of l-histidinol phosphate aminotransferase from Escherichia coli, as a complex with pyridoxamine-5'-phosphate (PMP) at 1.5 A resolution, as the internal aldimine with PLP, and in a covalent, tetrahedral complex consisting of PLP and l-histidinol phosphate attached to Lys214, both at 2.2 A resolution. This covalent complex resembles, in structural terms, the gem-diamine intermediate that is formed transiently during conversion of the internal to external aldimine.HisC is a dimeric enzyme with a mass of approximately 80 kDa. Like most PLP-dependent enzymes, each HisC monomer consists of two domains, a larger PLP-binding domain having an alpha/beta/alpha topology, and a smaller domain. An N-terminal arm contributes to the dimerization of the two monomers. The PLP-binding domain of HisC shows weak sequence similarity, but significant structural similarity with the PLP-binding domains of a number of PLP-dependent enzymes. Residues that interact with the PLP cofactor, including Tyr55, Asn157, Asp184, Tyr187, Ser213, Lys214 and Arg222, are conserved in the family of aspartate, tyrosine and histidinol phosphate aminotransferases. The imidazole ring of l-histidinol phosphate is bound, in part, through a hydrogen bond with Tyr110, a residue that is substituted by Phe in the broad substrate specific HisC enzymes from Zymomonas mobilis and Bacillus subtilis. Comparison of the structures of the HisC internal aldimine, the PMP complex and the HisC l-histidinol phosphate complex reveal minimal changes in protein or ligand structure. Proton transfer, required for conversion of the gem-diamine to the external aldimine, does not appear to be limited by the distance between substrate and lysine amino groups. We propose that the tetrahedral complex has resulted from non-productive binding of l-histidinol phosphate soaked into the HisC crystals, resulting in its inability to be converted to the external aldimine at the HisC active site.

The crystal structure of Escherichia coli MoeA, a protein from the molybdopterin synthesis pathway.

MoeA is involved in synthesis of the molybdopterin cofactor, although its function is not yet clearly defined. The three-dimensional structure of the Escherichia coli protein was solved at 2.2 A resolution. The locations of highly conserved residues among the prokaryotic and eukaryotic MoeA homologs identifies a cleft in the dimer interface as the likely functional site. Of the four domains of MoeA, domain 2 displays a novel fold and domains 1 and 4 each have only one known structural homolog. Domain 3, in contrast, is structurally similar to many other proteins. The protein that resembles domain 3 most closely is MogA, another protein required for molybdopterin cofactor synthesis. The overall similarity between MoeA and MogA, and the similarities in a constellation of residues that are strongly conserved in MoeA, suggests that these proteins bind similar ligands or substrates and may have similar functions.

Active site of chondroitin AC lyase revealed by the structure of enzyme-oligosaccharide complexes and mutagenesis.

The crystal structures of Flavobacterium heparinium chondroitin AC lyase (chondroitinase AC; EC 4.2.2.5) bound to dermatan sulfate hexasaccharide (DS(hexa)), tetrasaccharide (DS(tetra)), and hyaluronic acid tetrasaccharide (HA(tetra)) have been refined at 2.0, 2.0, and 2.1 A resolution, respectively. The structure of the Tyr234Phe mutant of AC lyase bound to a chondroitin sulfate tetrasaccharide (CS(tetra)) has also been determined to 2.3 A resolution. For each of these complexes, four (DS(hexa) and CS(tetra)) or two (DS(tetra) and HA(tetra)) ordered sugars are visible in electron density maps. The lyase AC DS(hexa) and CS(tetra) complexes reveal binding at four subsites, -2, -1, +1, and +2, within a narrow and shallow protein channel. We suggest that subsites -2 and -1 together represent the substrate recognition area, +1 is the catalytic subsite and +1 and +2 together represent the product release area. The putative catalytic site is located between the substrate recognition area and the product release area, carrying out catalysis at the +1 subsite. Four residues near the catalytic site, His225, Tyr234, Arg288, and Glu371 together form a catalytic tetrad. The mutations His225Ala, Tyr234Phe, Arg288Ala, and Arg292Ala, revealed residual activity for only the Arg292Ala mutant. Structural data indicate that Arg292 is primarily involved in recognition of the N-acetyl and sulfate moieties of galactosamine, but does not participate directly in catalysis. Candidates for the general base, removing the proton attached to C-5 of the glucuronic acid at the +1 subsite, are Tyr234, which could be transiently deprotonated during catalysis, or His225. Tyrosine 234 is a candidate to protonate the leaving group. Arginine 288 likely contributes to charge neutralization and stabilization of the enolate anion intermediate during catalysis.

Three-dimensional structure of 2-amino-3-ketobutyrate CoA ligase from Escherichia coli complexed with a PLP-substrate intermediate: inferred reaction mechanism.

2-Amino-3-ketobutyrate CoA ligase (KBL, EC 2.3.1.29) is a pyridoxal phosphate (PLP) dependent enzyme, which catalyzes the second reaction step on the main metabolic degradation pathway for threonine. It acts in concert with threonine dehydrogenase and converts 2-amino-3-ketobutyrate, the product of threonine dehydrogenation by the latter enzyme, with the participation of cofactor CoA, to glycine and acetyl-CoA. The enzyme has been well conserved during evolution, with 54% amino acid sequence identity between the Escherichia coli and human enzymes. We present the three-dimensional structure of E. coli KBL determined at 2.0 A resolution. KBL belongs to the alpha family of PLP-dependent enzymes, for which the prototypic member is aspartate aminotransferase. Its closest structural homologue is E. coli 8-amino-7-oxononanoate synthase. Like many other members of the alpha family, the functional form of KBL is a dimer, and one such dimer is found in the asymmetric unit in the crystal. There are two active sites per dimer, located at the dimer interface. Both monomers contribute side chains to each active/substrate binding site. Electron density maps indicated the presence in the crystal of the Schiff base intermediate of 2-amino-3-ketobutyrate and PLP, an external aldimine, which remained bound to KBL throughout the protein purification procedure. The observed interactions between the aldimine and the side chains in the substrate binding site explain the specificity for the substrate and provide the basis for a detailed proposal of the reaction mechanism of KBL. A putative binding site of the CoA cofactor was assigned, and implications for the cooperation with threonine dehydrogenase were considered.

Channeling efficiency in the bifunctional methylenetetrahydrofolate dehydrogenase/cyclohydrolase domain: the effects of site-directed mutagenesis of NADP binding residues.

The three-dimensional structure of the dehydrogenase-cyclohydrolase bifunctional domain of the human trifunctional enzyme indicates that Arg-173 and Ser-197 are within 3 A of the 2'-phosphate of bound NADP. Site-directed mutagenesis confirms that Arg-173 is essential for efficient binding and cannot be substituted by lysine. R173A and R173K have detectable dehydrogenase activity, but the K(m) values for NADP are increased by at least 500-fold. The S197A mutant has a K(m) for NADP that is only 20-fold higher than wild-type, indicating that it plays a supporting role. Forward and reverse cyclohydrolase activities of all the mutants were unchanged, except that the reverse cyclohydrolase activity of mutants that bind NADP poorly, or lack Ser-197, cannot be stimulated by 2',5'-ADP. The 50% channeling efficiency in the forward direction is not improved by the addition of exogenous NADPH and cannot be explained by premature dissociation of the dinucleotide from the ternary complex. As well, channeling is unaffected in mutants that exhibit a wide range of dinucleotide binding. Given that dinucleotide binding is unrelated to substrate channeling efficiency in the D/C domain, we propose that the difference in forward and reverse channeling efficiencies can be explained solely by the movement of the methenylH(4)folate between two overlapping subsites to which it has different binding affinities.

Crystallization and preliminary X-ray analysis of chondroitin sulfate ABC lyases I and II from Proteus vulgaris.

Chondroitin sulfate ABC lyases (E.C. 4.2.2.4) are broad-specificity glycosaminoglycan-degrading enzymes. Their preferred substrates are chondroitin sulfate and dermatan sulfate, which are broken down to short oligosaccharides. Proteus vulgaris produces two such lyases, ABC lyase I and II, with molecular weights of 112-113 kDa. Diffraction-quality crystals of both enzymes have been obtained by the hanging-drop vapour-diffusion method. ABC lyase I crystallizes in space group P2(1)2(1)2(1), with unit-cell parameters a = 49.3, b = 95.1, c = 230.0 A, Z = 4, and diffracts to 1.9 A resolution. Crystals of ABC lyase II belong to space group P1, with unit-cell parameters a = 64.2, b = 64.3, c = 142.1 A, alpha = 95.7, beta = 98. 1, gamma = 95.5 degrees, Z = 2; diffraction extends to at least 2.1 A.

Crystal structure of human grancalcin, a member of the penta-EF-hand protein family.

Grancalcin is a Ca(2+)-binding protein expressed at high level in neutrophils. It belongs to the PEF family, proteins containing five EF-hand motifs and which are known to associate with membranes in Ca(2+)-dependent manner. Prototypic members of this family are Ca(2+)-binding domains of calpain. Our recent finding that grancalcin interacts with L-plastin, a protein known to have actin bundling activity, suggests that grancalcin may play a role in regulation of adherence and migration of neutrophils. The structure of human grancalcin has been determined at 1.9 A resolution in the absence of calcium (R-factor of 0.212 and R-free of 0.249) and at 2. 5 A resolution in the presence of calcium (R-factor of 0.226 and R-free of 0.281). The molecule is predominantly alpha-helical: it contains eight alpha-helices and only two short stretches of two-stranded beta-sheets between the loops of paired EF-hands. Grancalcin forms dimers through the association of the unpaired EF5 hands in a manner similar to that observed in calpain, confirming this mode of association as a paradigm for the PEF family. Only one Ca(2+) was found per dimer under crystallization conditions that included CaCl(2). This cation binds to EF3 in one molecule, while this site in the second molecule of the dimer is unoccupied. This unoccupied site shows higher mobility. The structure determined in the presence of calcium, although does not represent a fully Ca(2+)-loaded form, suggests that calcium induces rather small conformational rearrangements. Comparison with calpain suggests further that the relatively small magnitude of conformational changes invoked by calcium alone may be a characteristic feature of the PEF family. Moreover, the largest differences are localized to the EF1, thus supporting the notion that calcium signaling occurs through this portion of the molecule and that it may involve the N-terminal Gly/Pro rich segment. Electrostatic potential distribution shows significant differences between grancalcin and calpain domain VI demonstrating their distinct character.

Structures of three inhibitor complexes provide insight into the reaction mechanism of the human methylenetetrahydrofolate dehydrogenase/cyclohydrolase.

Enzymes involved in tetrahydrofolate metabolism are of particular pharmaceutical interest, as their function is crucial for amino acid and DNA biosynthesis. The crystal structure of the human cytosolic methylenetetrahydrofolate dehydrogenase/cyclohydrolase (DC301) domain of a trifunctional enzyme has been determined previously with a bound NADP cofactor. While the substrate binding site was identified to be localized in a deep and rather hydrophobic cleft at the interface between two protein domains, the unambiguous assignment of catalytic residues was not possible. We succeeded in determining the crystal structures of three ternary DC301/NADP/inhibitor complexes. Investigation of these structures followed by site-directed mutagenesis studies allowed identification of the amino acids involved in catalysis by both enzyme activities. The inhibitors bind close to Lys56 and Tyr52, residues of a strictly conserved motif for active sites in dehydrogenases. While Lys56 is in a good position for chemical interaction with the substrate analogue, Tyr52 was found stacking against the inhibitors' aromatic rings and hence seems to be more important for proper positioning of the ligand than for catalysis. Also, Ser49 and/or Cys147 were found to possibly act as an activator for water in the cyclohydrolase step. These and the other residues (Gln100 and Asp125), with which contacts are made, are strictly conserved in THF dehydrogenases. On the basis of structural and mutagenesis data, we propose a reaction mechanism for both activities, the dehydrogenase and the cyclohydrolase.

Roles of individual EF-hands in the activation of m-calpain by calcium.

m-Calpain is a heterodimeric, cytosolic, thiol protease, which is activated by Ca(2+)-binding to EF-hands in the C-terminal domains of both subunits. There are four potential Ca(2+)-binding EF-hands in each subunit, but their relative affinities for Ca(2+) are not known. In the present study mutations were made in both subunits to reduce the Ca(2+)-binding affinity at one or more EF-hands in one or both subunits. X-ray crystallography of some of the mutated small subunits showed that Ca(2+) did not bind to the mutated EF-hands, but that its binding at other sites was not affected. The structures of the mutant small subunits in the presence of Ca(2+) were otherwise identical to that of the Ca(2+)-bound wild-type small subunit. In the whole enzyme the wild-type macroscopic Ca(2+) requirement (K(d)) was approx. 350 microM. The mutations did not affect the maximum specific activity of the enzyme, but caused increases in K(d), which were characteristic of each site. All the EF-hands could be mutated in various combinations without loss of activity, but preservation of at least one wild-type EF-hand 3 sequence was required to maintain K(d) values lower than 1 mM. The results suggest that all the EF-hands can contribute co-operatively to calpain activation, but that EF-hand 3, in both subunits, has the highest intrinsic affinity for Ca(2+) and provides the major driving force for conformational change.

Crystallization and preliminary X-ray analysis of human grancalcin, a novel cytosolic Ca2+-binding protein present in leukocytes.

Recombinant human grancalcin, a calcium-binding protein from leukocytes, has been crystallized in the presence or absence of Ca(2+) by the vapor-diffusion method. Two crystal forms of apo grancalcin were obtained: space group P2(1), with unit-cell parameters a = 48.4, b = 81.1, c = 46.6 A, beta = 111.3 degrees, diffracting to 1.9 A, and space group C2, with unit-cell parameters a = 97.0, b = 51.9, c = 75.9 A, beta = 108.5 degrees, diffracting to 2.4 A. Crystals were also grown in the presence of 5 mM Ca(2+). They also belong to space group C2, with unit-cell parameters a = 97.4, b = 50.3, c = 77.6 A, beta = 108.2 degrees, which are very similar to the second apo grancalcin form. These crystals diffract to 2.5 A.

Design of P1' and P3' residues of trivalent thrombin inhibitors and their crystal structures.

Synthetic bivalent thrombin inhibitors comprise an active site blocking segment, a fibrinogen recognition exosite blocking segment, and a linker connecting these segments. Possible nonpolar interactions of the P1' and P3' residues of the linker with thrombin S1' and S3' subsites, respectively, were identified using the "Methyl Scan" method [Slon-Usakiewicz et al. (1997) Biochemistry 36, 13494-13502]. A series of inhibitors (4-tert-butylbenzenesulfonyl)-Arg-(D-pipecolic acid)-Xaa-Gly-Yaa-Gly-betaAla-Asp-Tyr-Glu-Pro-Ile-Pro-Glu-Glu-Ala- (be ta-cyclohexylalanine)-(D-Glu)-OH, in which nonpolar P1' residue Xaa or P3' residue Yaa was incorporated, were designed and improved the affinity to thrombin. Substitution of the P3' residue with D-phenylglycine or D-Phe improved the K(i) value to (9.5 +/- 0.6) x 10(-14) or 1.3 +/- 0.5 x 10(-13) M, respectively, compared to that of a reference inhibitor with Gly residues at Xaa and Yaa residues (K(i) = (2.4 +/- 0.5) x 10(-11) M). Similarly, substitution of the P1' residue with L-norleucine or L-beta-(2-thienyl)alanine lowered the K(i) values to (8.2 +/- 0.6) x 10(-14) or (5.1 +/- 0.4) x 10(-14) M, respectively. The linker Gly-Gly-Gly-betaAla of the inhibitors in the previous sentence was simplified with 12-aminododecanoic acid, resulting in further improvement of the K(i) values to (3.8 +/- 0.6) x 10(-14) or (1.7 +/- 0.4) x 10(-14) M, respectively. These K(i) values are equivalent to that of natural hirudin (2.2 x 10(-14) M), yet the size of the synthetic inhibitors (2 kD) is only one-third that of hirudin (7 kD). Two inhibitors, with L-norleucine or L-beta-(2-thienyl)alanine at the P1' residue and the improved linker of 12-aminododecanoic acid, were crystallized in complex with human alpha-thrombin. The crystal structures of these complexes were solved and refined to 2.1 A resolution. The Lys(60F) side chain of thrombin moved significantly and formed a large nonpolar S1' subsite to accommodate the bulky P1' residue.

Crystal structure of human procathepsin X: a cysteine protease with the proregion covalently linked to the active site cysteine.

Human cathepsin X is one of many proteins discovered in recent years through the mining of sequence databases. Its sequence shows clear homology to cysteine proteases from the papain family, containing the characteristic residue patterns, including the active site. However, the proregion of cathepsin X is only 38 residues long, the shortest among papain-like enzymes, and the cathepsin X sequence has an atypical insertion in the regions proximal to the active site. This protein was recently expressed and partially characterized biochemically. Unlike most other cysteine proteases from the papain family, procathepsin X is incapable of autoprocessing in vitro but can be processed under reducing conditions by exogenous cathepsin L. Atypically, the mature enzyme is primarily a carboxypeptidase and has extremely poor endopeptidase activity. We have determined the three-dimensional structure of the procathepsin X at 1.7 A resolution. The overall structure of the mature enzyme is characteristic for enzymes of the papain superfamily, but contains several novel features. Most interestingly, the short proregion binds to the enzyme with the aid of a covalent bond between the cysteine residue in the proregion (Cys10p) and the active site cysteine residue (Cys31). This is the first example of a zymogen in which the inhibition of enzyme's proteolytic activity by the proregion is achieved through a reversible covalent modification of the active site nucleophile. Such mode of binding requires less contact area between the proregion and the enzyme than observed in other procathepsins, and no auxiliary binding site on the enzyme surface is used. A three-residue insertion in a highly conserved region, just prior to the active site cysteine residue, confers a significantly different shape on the S' subsites, compared to other proteases from papain family. The 3D structure provides an explanation for the rather unusual carboxypeptidase activity of this enzyme and confirms the predictions based on homology modeling. Another long insertion in the cathepsin X amino acid sequence forms a beta-hairpin pointing away from the active site. This insertion, thought to be an equivalent of cathepsin B occluding loop, is located on the side of the protein, distant from the substrate binding site.

Crystal structure of chondroitinase B from Flavobacterium heparinum and its complex with a disaccharide product at 1.7 A resolution.

Glycosaminoglycans (GAGs) are a family of acidic heteropolysaccharides, including such molecules as chondroitin sulfate, dermatan sulfate, heparin and keratan sulfate. Cleavage of the O-glycosidic bond within GAGs can be accomplished by hydrolases as well as lyases, yielding disaccharide and oligosaccharide products. We have determined the crystal structure of chondroitinase B, a glycosaminoglycan lyase from Flavobacterium heparinum, as well as its complex with a dermatan sulfate disaccharide product, both at 1.7 A resolution. Chondroitinase B adopts the right-handed parallel beta-helix fold, found originally in pectate lyase and subsequently in several polysaccharide lyases and hydrolases. Sequence homology between chondroitinase B and a mannuronate lyase from Pseudomonas sp. suggests this protein also adopts the beta-helix fold. Binding of the disaccharide product occurs within a positively charged cleft formed by loops extending from the surface of the beta-helix. Amino acid residues responsible for recognition of the disaccharide, as well as potential catalytic residues, have been identified. Two arginine residues, Arg318 and Arg364, are found to interact with the sulfate group attached to O-4 of N-acetylgalactosamine. Cleavage of dermatan sulfate likely occurs at the reducing end of the disaccharide, with Glu333 possibly acting as the general base.

Structure and conformational flexibility of Candida rugosa lipase.

Three-dimensional structures of a number of lipases determined in the past decade have provided a solid structural foundation for our understanding of lipase function. The structural studies of Candida rugosa lipase summarized here have addressed many facets of interfacial catalysis. These studies have revealed a fold and catalytic site common to other lipases. Different conformations likely to correlate with interfacial activation of the enzyme were observed in different crystal forms. The structures of enzyme-inhibitor complexes have identified the binding site for the scissile fatty acyl chain, provided the basis for molecular modeling of triglyceride binding and provided insight into the structural basis of the common enantiopreferences shown by lipases.

Crystal structure of chondroitin AC lyase, a representative of a family of glycosaminoglycan degrading enzymes.

Glycosaminoglycans (GAGs), highly sulfated polymers built of hexosamine-uronic acid disaccharide units, are major components of the extracellular matrix, mostly in the form of proteoglycans. They interact with a large array of proteins, in particular of the blood coagulation cascade. Degradation of GAGs in mammalian systems occurs by the action of GAG hydrolases. Bacteria express a large number of GAG-degrading lyases that break the hexosamine-uronic acid bond to create an unsaturated sugar ring. Flavobacterium heparinum produces at least five GAG lyases of different specificity. Chondroitin AC lyase (chondroitinase AC, 75 kDa) is highly active toward chondroitin 4-sulfate and chondroitin-6 sulfate. Its crystal structure has been determined to 1.9 A resolution. The enzyme is composed of two domains. The N-terminal domain of approximately 300 residues contains mostly alpha-helices which form a doubly-layered horseshoe (a subset of the (alpha/alpha)6 toroidal topology). The approximately 370 residues long C-terminal domain is made of beta-strands arranged in a four layered beta-sheet sandwich, with the first two sheets having nine strands each. This fold is novel and has no counterpart in full among known structures. The sequence of chondroitinase AC shows low level of homology to several hyaluronate lyases, which likely share its fold. The shape of the molecule, distribution of electrostatic potential, the pattern of conservation of the amino acids and the results of mutagenesis of hyaluronate lyases, indicate that the enzymatic activity resides primarily within the N-terminal domain. The most likely candidate for the catalytic base is His225. Other residues involved in catalysis and/or substrate binding are Arg288, Arg292, Lys298 and Lys299.

The occluding loop in cathepsin B defines the pH dependence of inhibition by its propeptide.

Papain-like proenzymes are prone to autoprocess under acidic pH conditions. Similarly, peptides derived from the proregion of cathepsin B are potent pH-dependent inhibitors of that enzyme; i.e., at pH 6.0 the inhibition of human cathepsin B by its propeptide is defined by slow binding kinetics with a Ki of 3.7 nM and at pH 4.0 by classical kinetics with a Ki of 82 nM. This pH dependency is essentially eliminated either by the removal of a portion of the enzyme's occluding loop through deletion mutagenesis or by the mutation of either residue Asp22 or His110 to alanine; e.g., the mutant enzyme His110Ala is inhibited by its propeptide with Ki's of 2.0 +/- 0.3 nM at pH 4.0 and 1.1 +/- 0.2 nM at pH 6.0. For the His110Ala mutant the inhibition also displays slow binding kinetics at both pH 4.0 and pH 6.0. As shown by the crystal structure of mature cathepsin B [Musil, D., et al. (1991) EMBO J. 10, 2321-2330] Asp22 and His110 form a salt bridge in the mature enzyme, and it has been shown that this bridge stabilizes the occluding loop in its closed position [Nägler, D. K., et al. (1997) Biochemistry 36, 12608-12615]. Thus the pH dependency of propeptide binding can be explained on the basis of a competitive binding between the occluding loop and the propeptide. At low pH, when the Asp22-His110 pair forms a salt bridge stabilizing the occluding loop in its closed conformation, the loop more effectively competes with the propeptide than at higher pH where deprotonation of His110 and the concomitant destruction of the Asp22-His110 salt bridge results in a destabilization of the closed form of the loop. The rate of autocatalytic processing of procathepsin B to cathepsin B correlates with the affinity of the enzyme for its propeptide rather than with its catalytic activity, thus suggesting a possible influence of occluding loop stability on the rate of processing.