Thermodynamic and circular dichroism studies differentiate inhibitor interactions with the stromelysin S(1)-S(3) and S(')(1)-S(')(3) subsites.

Interactions of stromelysin with a series of inhibitors representative of three chemical templates with distinct binding modes were examined. Unfolding temperatures for inhibitor complexes were 10 degrees C to 15 degrees C greater than for apo stromelysin. Minor changes in ellipticity in the far-UV CD spectra of complexes indicated that ligand-induced conformational changes were localized to the binding site and did not involve gross changes in protein folding. Isothermal titrating calorimetry of thiadiazole-containing inhibitors, which bind in the S(1)-S(3) subsites of stromelysin, indicated that the binding interaction was exothermic and only slightly favorable entropically. Near-UV CD spectra showed large positive ellipticity increases from 250 to 300 nm, consistent with an interaction between the benzene ring of the inhibitor and stromelysin residues Tyr155 and Tyr168. Interactions between stromelysin and amide-hydroxamate ligands, which bind in the S(')(1)-S(')(3) subsites, were found to be both enthalpically and entropically driven. Binding of this class of ligands resulted in modest negative ellipticity changes at 260-285 nm and positive increases at 292 nm. Stromelysin complexed to a lactam-hydroxamate inhibitor with structure extending into both the S(1)-S(3) and S(')(1)-S(')(3) subsites showed increased ellipticity at 245 nm and negative changes at 260-285 and 295 nm.     

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.     

Crystallographic and biochemical investigations of kumamolisin-As, a serine-carboxyl peptidase with collagenase activity

Kumamolisin-As (previously called ScpA) is the first known example of a collagenase from the sedolisin family (MEROPS S53). This enzyme is active at low pH and in elevated temperatures. In this study that used x-ray crystallographic and biochemical methods, we investigated the structural basis of the preference of this enzyme for collagen and the importance of a glutamate residue in the unique catalytic triad (Ser(278)-Glu(78)-Asp(82)) for enzymatic activity. Crystal structures of the uninhibited enzyme and its complex with a covalently bound inhibitor, N-acetyl-isoleucyl-prolyl-phenylalaninal, showed the occurrence of a narrow S2 pocket and a groove that encompasses the active site and is rich in negative charges. Limited endoproteolysis studies of bovine type-I collagen as well as kinetic studies using peptide libraries randomized at P1 and P1', showed very strong preference for arginine at the P1 position, which correlated very well with the presence of a negatively charged residue in the S1 pocket of the enzyme. All of these features, together with those predicted through comparisons with fiddler crab collagenase, a serine peptidase, rationalize the enzyme's preference for collagen. A comparison of the Arrhenius plots of the activities of kumamolisin-As with either collagen or peptides as substrates suggests that collagen should be relaxed before proteolysis can occur. The E78H mutant, in which the catalytic triad was engineered to resemble that of subtilisin, showed only 0.01% activity of the wild-type enzyme, and its structure revealed that Ser(278), His(78), and Asp(82) do not interact with each other; thus, the canonical catalytic triad is disrupted.     

The crystal structures of recombinant glycosylated human renin alone and in complex with a transition state analog inhibitor

Recombinant human glycosylated renin has been crystallized in complex with CGP 38'560, a transition state analog inhibitor (IC50 = 2 x 10(-9) M), in a tetragonal crystal form. The structure has been determined to a resolution of 2.4 A and refined to a crystallographic Rfactor of 17.6%. It reveals the conformation of the inhibitor as well as its interactions with the enzyme active site. The active site is a deep cleft between the N- and the C-terminal domains to which the inhibitor binds in an extended conformation filling the S4 to S2' pockets. The structure of the complex is compared with that of the related uninhibited enzyme pepsin. Significant changes in the relative orientation of the N- and C-terminal domains are observed. In the inhibited renin structure the C-terminal loop segments forming the active site are closer to those from the N-terminal domain than in the related "open" pepsin structure. In addition, the structure of uninhibited glycosylated renin has been determined at 2.8 A resolution from a cubic crystal form with two renin molecules in the asymmetric unit. The two independent renin molecules show different conformations with respect to the relative orientation of their N- and C-terminal domains; one molecule is found in the "closed inhibited" conformation, the other in the "open uninhibited" conformation.     

Substrate-related potent inhibitors of brain metalloendopeptidase

Rat brain metalloendopeptidase (EC 3.4.24.15) generates Leu- and Met-enkephalin from several larger opioid peptides and is capable of degrading a number of neuropeptides. Substrate-related N-(1-carboxy-3-phenylpropyl) peptide derivatives were synthesized and tested for enzyme inhibition. The best of these derivatives, N-[1(RS)-carboxy-3-phenylpropyl]-Ala-Ala-Tyr-p-aminobenzoate, inhibited the enzyme in a competitive manner with a Ki of 16 nM. The data indicate that the carboxyl group of the N-(1-carboxy-3-phenylpropyl) moiety coordinates with the active site zinc atom and that the remaining part of the inhibitor is necessary for interaction with the substrate recognition site of the enzyme. Replacement of the 1-carboxy-3-phenylpropyl group by a carboxymethyl group decreased the inhibitory potency by more than 3 orders of magnitude, emphasizing the importance of the hydrophobic phenyl group for inhibitor binding to a hydrophobic pocket at the S1 subsite. Replacement of the Tyr residue by an Ala residue decreased the inhibitory potency by more than 20-fold. Changes in the structure of the residue interacting with the S1' subsite could cause a more than 60-fold change in inhibition. The inhibitors were either ineffective or only weakly inhibitory against membrane-bound metalloendopeptidase ("enkephalinase", EC 3.4.24.11), an enzyme highly active in rabbit kidney but also present in brain. The data indicate the presence of an extended binding site in the enzyme with residues interacting with S1, S1', and S3' subsites largely determining inhibitor binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Conserved water mediated recognition and the dynamics of active site Cys 331 and Tyr 411 in hydrated structure of human IMPDH‐II

Inosine monophosphate dehydrogenase (IMPDH) of human is involved in GMP biosynthesis pathway, increased level of IMPDH‐II (an isoform of enzyme) activity have found in leukemic and sarcoma cells. Modeling and extensive molecular dynamics simulation (15 ns) studies of IMPDH‐II (1B3O PDB structure) have indicated the intricate involvement of four conserved water molecules (W 1, W 2, W 3, and W 4) in the conformational transition or the mobilities of “flap” (residues 400–450) and “loop” (residues 325–342) regions in enzyme. The stabilization of active site residues Asn 303, Gly 324, Ser 329, Cys 331, Asp 364, and Tyr 411 through variable H‐bonding coordination from the conserved water molecular center seems interesting in the uninhibited hydrated form of human IMPDH‐II structures. This conformational transition or the flexibility of mobile regions, water molecular recognition to active site residues Cys 331 and Tyr 411, and the presence of a hydrophilic cavity ～540 ų (enclaved by the loop and flap region) near the C‐terminal surface of this enzyme may explore a rational hope toward the water mimic inhibitor or anticancer agent design for human. Copyright © 2010 John Wiley & Sons, Ltd.     

Residues Tyr253 and Glu255 in strand 3 of beta-sheet C of antithrombin are key determinants of an exosite made accessible by heparin activation to promote rapid inhibition of factors Xa and IXa

We previously showed that conformational activation of the anticoagulant serpin, antithrombin, by heparin generates new exosites in strand 3 of beta-sheet C, which promote the reaction of the inhibitor with the target proteases, factor Xa and factor IXa. To determine which residues comprise the exosites, we mutated strand 3C residues that are conserved in all vertebrate antithrombins. Combined mutations of the three conserved surface-accessible residues, Tyr253,Glu255, and Lys257, or of just Tyr253 and Glu255, but not any of these residues alone, was sufficient to reproduce the exosite defects of a strand 3C antithrombin-alpha1-proteinase inhibitor chimera in reactions of the heparin-activated variants with both factor Xa and factor IXa. Importantly, the exosite-defective antithrombins bound heparin with nearly wild-type affinities, and the heparin-activated mutants showed near normal reactivities with thrombin, a protease that does not utilize the exosite. Mutation of the conserved but partially buried strand 3C residue, Gln254, the reactive loop P6' residue, Arg399, which interacts with Glu255, or a residue proposed to constitute the exosite from modeling studies, Glu237, all produced minimal effects on antithrombin reactivity with thrombin, factor Xa, and factor IXa in the absence or presence of heparin. Together, these results indicate that Tyr253 and Glu255 are key exosite determinants responsible for promoting the reactions of conformationally activated antithrombin with both factor Xa and factor IXa.     

The catalytic reaction and inhibition mechanism of Drosophila alcohol dehydrogenase: observation of an enzyme-bound NAD-ketone adduct at 1.4 A resolution by X-ray crystallography.

Drosophila alcohol dehydrogenase (DADH) is an NAD+-dependent enzyme that catalyzes the oxidation of alcohols to aldehydes/ketones. DADH is the member of the short-chain dehydrogenases/reductases family (SDR) for which the largest amount of biochemical data has been gathered during the last three decades. The crystal structures of one binary form (NAD+) and three ternary complexes with NAD+.acetone, NAD+.3-pentanone and NAD+.cyclohexanone were solved at 2.4, 2.2, 1. 4 and 1.6 A resolution, respectively. From the molecular interactions observed, the reaction mechanism could be inferred. The structure of DADH undergoes a conformational change in order to bind the coenzyme. Furthermore, upon binding of the ketone, a region that was disordered in the apo form (186-191) gets stabilized and closes the active site cavity by creating either a small helix (NAD+. acetone, NAD+.3-pentanone) or an ordered loop (NAD+.cyclohexanone). The active site pocket comprises a hydrophobic bifurcated cavity which explains why the enzyme is more efficient in oxidizing secondary aliphatic alcohols (preferably R form) than primary ones. Difference Fourier maps showed that the ketone inhibitor molecule has undergone a covalent reaction with the coenzyme in all three ternary complexes. Due to the presence of the positively charged ring of the coenzyme (NAD+) and the residue Lys155, the amino acid Tyr151 is in its deprotonated (tyrosinate) state at physiological pH. Tyr151 can subtract a proton from the enolic form of the ketone and catalyze a nucleophilic attack of the Calphaatom to the C4 position of the coenzyme creating an NAD-ketone adduct. The binding of these NAD-ketone adducts to DADH accounts for the inactivation of the enzyme. The catalytic reaction proceeds in a similar way, involving the same amino acids as in the formation of the NAD-ketone adduct. The p Kavalue of 9-9.5 obtained by kinetic measurements on apo DADH can be assigned to a protonated Tyr151 which is converted to an unprotonated tyrosinate (p Ka7.6) by the influence of the positively charged nicotinamide ring in the binary enzyme-NAD+form. pH independence during the release of NADH from the binary complex enzyme-NADH can be explained by either a lack of electrostatic interaction between the coenzyme and Tyr151 or an apparent p Kavalue for this residue higher than 10.0.     

Stromelysin-1: three-dimensional structure of the inhibited catalytic domain and of the C-truncated proenzyme.

The proteolytic enzyme stromelysin-1 is a member of the family of matrix metalloproteinases and is believed to play a role in pathological conditions such as arthritis and tumor invasion. Stromelysin-1 is synthesized as a pro-enzyme that is activated by removal of an N-terminal prodomain. The active enzyme contains a catalytic domain and a C-terminal hemopexin domain believed to participate in macromolecular substrate recognition. We have determined the three-dimensional structures of both a C-truncated form of the proenzyme and an inhibited complex of the catalytic domain by X-ray diffraction analysis. The catalytic core is very similar in the two forms and is similar to the homologous domain in fibroblast and neutrophil collagenases, as well as to the stromelysin structure determined by NMR. The prodomain is a separate folding unit containing three alpha-helices and an extended peptide that lies in the active site of the enzyme. Surprisingly, the amino-to-carboxyl direction of this peptide chain is opposite to that adopted by the inhibitor and by previously reported inhibitors of collagenase. Comparison of the active site of stromelysin with that of thermolysin reveals that most of the residues proposed to play significant roles in the enzymatic mechanism of thermolysin have equivalents in stromelysin, but that three residues implicated in the catalytic mechanism of thermolysin are not represented in stromelysin.     

An unusual orientation for Tyr75 in the active site of the aspartic proteinase from Saccharomyces cerevisiae

The structures of the native Saccharomyces cerevisiae proteinase A have been solved by molecular replacement in the monoclinic and trigonal crystal forms and refined at 2.6-2.7A resolution. These structures agree overall with those of other uninhibited aspartic proteinases. However, an unusual orientation for the side chain of Tyr75, a conserved residue on the flexible "flap" that covers the active site and is important for the activity of these enzymes, was found in the trigonal crystals. A similar conformation of Tyr75 occupying the S1 substrate-binding pocket was previously reported only for chymosin (where it was interpreted as representing a "self-inhibited" state of the enzyme), but for no other aspartic proteinases. Since this orientation of Tyr75 has now been seen in the structures of two members of the family of aspartic proteinases, it might indicate that the placement of that residue in the S1 substrate-binding pocket might have some functional significance, analogous to what was seen for self-inhibited structures of serine proteinases.     

High-resolution X-ray diffraction study of the complex between endothiapepsin and an oligopeptide inhibitor: the analysis of the inhibitor binding and description of the rigid body shift in the enzyme.

The conformation of the synthetic renin inhibitor CP-69,799, bound to the active site of the fungal aspartic proteinase endothiapepsin (EC 3.4.23.6), has been determined by X-ray diffraction at 1.8 A resolution and refined to the crystallographic R factor of 16%. CP-69,799 is an oligopeptide transition--state analogue inhibitor that contains a new dipeptide isostere at the P1-P1' position. This dipeptide isostere is a nitrogen analogue of the well-explored hydroxyethylene dipeptide isostere, wherein the tetrahedral P1' C alpha atom has been replaced by trigonal nitrogen. The inhibitor binds in the extended conformation, filling S4 to S3' pockets, with hydroxyl group of the P1 residue positioned symmetrically between the two catalytic aspartates of the enzyme. Interactions between the inhibitor and the enzyme include 12 hydrogen bonds and extensive van der Waals contacts in all the pockets, except for S3'. The crystal structure reveals a bifurcated orientation of the P2 histidine side chain and an interesting relative rotation of the P3 phenyl ring to accommodate the cyclohexyl side chain at P1. The binding of the inhibitor to the enzyme, while producing no large distortions in the enzyme active site cleft, results in small but significant change in the relative orientation of the two endothiapepsin domains. This structural change may represent the action effected by the proteinase as it distorts its substrate towards the transition state for proteolytic cleavage.     

Bioactive conformation of a potent stromelysin inhibitor determined by X-nucleus filtered and multidimensional NMR spectroscopy

The biologically active conformation of a novel, very potent, nonpeptidic stromelysin inhibitor was determined by X-nucleus filtered and multidimensional NMR spectroscopy. This bound conformer was subsequently docked into the stromelysin catalytic domain (SCD) using intermolecular distance constraints derived from NOE data. The complex showed the S1′ pocket of stromelysin to be the major site of enzyme-inhibitor interaction with other portions of the inhibitor spanning the S2′ and S1 binding sites. Theoretical predictions of SCD-inhibitor binding from molecular modeling studies were consistent with the NMR data. Comparison of modeled enzyme-inhibitor complexes for stromelysin and collagenase revealed an alternate binding mode for the inhibitor in collagenase, suggesting a similar binding interaction might also be possible for stromelysin. The NMR results, however, revealed a single SCD-inhibitor binding mode and provided a structural template for the design of more potent stromelysin inhibitors.     

Solution structures of stromelysin complexed to thiadiazole inhibitors.

Unregulated or overexpressed matrix metalloproteinases (MMPs), including stromelysin, collagenase, and gelatinase. have been implicated in several pathological conditions including arthritis and cancer. Small-molecule MMP inhibitors may have therapeutic value in the treatment of these diseases. In this regard, the solution structures of two stromelysin/ inhibitor complexes have been investigated using 1H, 13C, and 15N NMR spectroscopy. Both-inhibitors are members of a novel class of matrix metalloproteinase inhibitor that contain a thiadiazole group and that interact with stromelysin in a manner distinct from other classes of inhibitors. The inhibitors coordinate the catalytic zinc atom through their exocyclic sulfur atom, with the remainder of the ligand extending into the S1-S3 side of the active site. The binding of inhibitor containing a protonated or fluorinated aromatic ring was investigated using 1H and 19F NMR spectroscopy. The fluorinated ring was found to have a reduced ring-flip rate compared to the protonated version. A strong, coplanar interaction between the fluorinated ring of the inhibitor and the aromatic ring of Tyr155 is proposed to account for the reduced ring-flip rate and for the increase in binding affinity observed for the fluorinated inhibitor compared to the protonated inhibitor. Binding interactions observed for the thiadiazole class of ligands have implications for the design of matrix metalloproteinase inhibitors.     

Analysis of the binding of hydroxamic acid and carboxylic acid inhibitors to the stromelysin-1 (matrix metalloproteinase-3) catalytic domain by isothermal titration calorimetry.

Matrix metalloproteinases (MMPs) are implicated in diseases such as arthritis and cancer. Among these enzymes, stromelysin-1 can also activate the proenzymes of other MMPs, making it an attractive target for pharmaceutical design. Isothermal titration calorimetry (ITC) was used to analyze the binding of three inhibitors to the stromelysin catalytic domain (SCD). One inhibitor (Galardin) uses a hydroxamic acid group (pK(a) congruent with 8.7) to bind the active site zinc; the others (PD180557 and PD166793) use a carboxylic acid group (pK(a) congruent with 4.7). Binding affinity increased dramatically as the pH was decreased over the range 5.5-7.5. Experiments carried out at pH 6.7 in several different buffers revealed that approximately one and two protons are transferred to the enzyme-inhibitor complexes for the hydroxamic and carboxylic acid inhibitors, respectively. This suggests that both classes of inhibitors bind in the protonated state, and that one amino acid residue of the enzyme also becomes protonated upon binding. Similar experiments carried out with the H224N mutant gave strong evidence that this residue is histidine 224. DeltaG, DeltaH, DeltaS, and DeltaC(p) were determined for the three inhibitors at pH 6.7, and DeltaC(p) was used to obtain estimates of the solvational, translational, and conformational components of the entropy term. The results suggest that: (1) a polar group at the P1 position can contribute a large favorable enthalpy, (2) a hydrophobic group at P2' can contribute a favorable entropy of desolvation, and (3) P1' substituents of certain sizes may trigger an entropically unfavorable conformational change in the enzyme upon binding. These findings illustrate the value of complete thermodynamic profiles generated by ITC in discovering binding interactions that might go undetected when relying on binding affinities alone.     

Crystal structure of Bacillus sp. GL1 xanthan lyase complexed with a substrate: insights into the enzyme reaction mechanism

Bacillus sp. GL1 xanthan lyase, a member of polysaccharide lyase family 8 (PL-8), acts exolytically on the side-chains of pentasaccharide-repeating polysaccharide xanthan and cleaves the glycosidic bond between glucuronic acid (GlcUA) and pyruvylated mannose (PyrMan) through a beta-elimination reaction. To clarify the enzyme reaction mechanism, i.e. its substrate recognition and catalytic reaction, we determined crystal structures of a mutant enzyme, N194A, in complexes with the product (PyrMan) and a substrate (pentasacharide) and in a ligand-free form at 1.8, 2.1, and 2.3A resolution. Based on the structures of the mutant in complexes with the product and substrate, we found that xanthan lyase recognized the PyrMan residue at subsite -1 and the GlcUA residue at +1 on the xanthan side-chain and underwent little interaction with the main chain of the polysaccharide. The structure of the mutant-substrate complex also showed that the hydroxyl group of Tyr255 was close to both the C-5 atom of the GlcUA residue and the oxygen atom of the glycosidic bond to be cleaved, suggesting that Tyr255 likely acts as a general base that extracts the proton from C-5 of the GlcUA residue and as a general acid that donates the proton to the glycosidic bond. A structural comparison of catalytic centers of PL-8 lyases indicated that the catalytic reaction mechanism is shared by all members of the family PL-8, while the substrate recognition mechanism differs.     

SNAP-25 substrate peptide (residues 180-183) binds to but bypasses cleavage by catalytically active Clostridium botulinum neurotoxin E

Clostridium botulinum neurotoxins are the most potent toxins to humans. The recognition and cleavage of SNAREs are prime evente in exhibiting their toxicity. We report here the crystal structure of the catalytically active full-length botulinum serotype E catalytic domain (BoNT E) in complex with SNAP-25 (a SNARE protein) substrate peptide Arg(180)-Ile(181)-Met(182)-Glu(183) (P1-P3'). It is remarkable that the peptide spanning the scissile bond binds to but bypasses cleavage by the enzyme and inhibits the catalysis fairly with K(i) approximately 69 microm. The inhibitory peptide occupies the active site of BoNT E and shows well defined electron density. The catalytic zinc and the conserved key residue Tyr(350) of the enzyme facilitate the docking of Arg(180) (P1) by interacting with its carbonyl oxygen that displaces the nucleophilic water. The general base Glu(212) side chain interacts with the main chain amino group of P1 and P1'. Conserved Arg(347) of BoNT E stabilizes the proper docking of the Ile(181) (P1') main chain, whereas the hydrophobic pockets stabilize the side chains of Ile(181) (P1') and Met(182) (P2'), and the 250 loop stabilizes Glu(183) (P3'). Structural and functional analysis revealed an important role for the P1' residue and S1' pocket in driving substrate recognition and docking at the active site. This study is the first of its kind and rationalizes the substrate cleavage strategy of BoNT E. Also, our complex structure opens up an excellent opportunity of structure-based drug design for this fast acting and extremely toxic high priority BoNT E.     

Maintenance of alpha-helical structures by phenyl rings in the active-site tyrosine triad contributes to catalysis and stability of ketosteroid isomerase from Pseudomonas putida biotype B.

Ketosteroid isomerase (KSI) from Pseudomonas putida biotype B is a homodimeric enzyme catalyzing an allylic rearrangement of Delta5-3-ketosteroids at rates comparable with the diffusion-controlled limit. The tyrosine triad (Tyr14.Tyr55.Tyr30) forming a hydrogen-bond network in the apolar active site of KSI has been characterized in an effort to identify the roles of the phenyl rings in catalysis, stability, and unfolding of the enzyme. The replacement of Tyr14, a catalytic residue, with serine resulted in a 33-fold decrease of kcat, while the replacements of Tyr30 and Tyr55 with serine decreased kcat by 4- and 51-fold, respectively. The large decrease of kcat for Y55S could be due to the structural perturbation of alpha-helix A3, which results in the reorientation of the active-site residues as judged by the crystal structure of Y55S determined at 2.2 A resolution. Consistent with the analysis of the Y55S crystal structure, the far-UV circular dichroism spectra of Y14S, Y30S, and Y55S indicated that the elimination of the phenyl ring of the tyrosine reduced significantly the content of alpha-helices. Urea-induced equilibrium unfolding experiments revealed that the DeltaG(U)H2O values of Y14S, Y30S, and Y55S were significantly decreased by 11.9, 13.7, and 9.5 kcal/mol, respectively, as compared with that of the wild type. A characterization of the unfolding kinetics based on PhiU-value analysis indicates that the interactions mediated by the tyrosine triad in the native state are very resistant to unfolding. Taken together, our results demonstrate that the internal packing by the phenyl rings in the active-site tyrosine triad contributes to the conformational stability and catalytic activity of KSI by maintaining the structural integrity of the alpha-helices.     

The catalytic power of ketosteroid isomerase investigated by computer simulation

Ketosteroid isomerase (KSI) catalyzes the isomerization of Delta(5)-3-ketosteroids and Delta(4)-3-ketosteroids at very high rates. Here we examine the principles underlying the catalytic efficiency of KSI by computer simulations using the empirical valence bond method in combination with molecular dynamics free energy perturbation simulations. The simulations reproduce available kinetic and structural data very well and allow us to examine several features of the catalytic mechanism in detail. It is found that about 60% of the rate enhancement is due to stabilization of the negatively charged dienolate intermediate by hydrogen bonding. The critical H-bond between Tyr16 and the intermediate is found to be a normal ionic H-bond with the preferred proton location on the tyrosine residue. The remaining 40% of the catalytic effect originates from a reduction of the reorganization energy of the reaction. The possibility of an active site water molecule occupying the empty cavity adjacent to the catalytic base (Asp40) is also addressed. The existence of such a water molecule could explain how the enzyme manages to maintain a low pK(a) for the general base residue.     

Pivotal molecular determinants of peptidic and collagen triple helicase activities reside in the S3' subsite of matrix metalloproteinase 8 (MMP-8): the role of hydrogen bonding potential of ASN188 and TYR189 and the connecting cis bond

The mechanism of triple helical collagen unwinding and cleavage by collagenases in the matrix metalloproteinase (MMP) family is complex and remains enigmatic. Recent reports show that triple helicase activity is initiated by the hemopexin C domain of membrane type 1-MMP, whereas catalytically inactive full-length interstitial collagenase (MMP-1) exhibits full triple helicase functionality pointing to active site determinants that are needed to complete the triple helicase mechanism. In MMP-8, the neutrophil collagenase, a conserved Gly at the S(3)' substrate specificity subsite is replaced by Asn(188) that forms a highly unusual cis bond with Tyr(189), a conserved active site residue in the collagenases. Only in MMP-1 is the S(3)' Gly also replaced, and there too a cis configured Glu-Tyr occurs. Thus, this high energy peptide bond coupled to the canonical Tyr may be important in the collagenolytic process. In a systematic mutagenesis investigation of the MMP-8 S(3)' subsite we found that introducing an S(3)' Gly(188) into MMP-8 reduced collagenolytic efficiency by approximately 30% with a corresponding reduction in cleavage of a synthetic peptide fluorescence resonance energy transfer substrate analogue of the alpha2(I) collagen chain cleavage site. The substitution of Asn(188) to Leu, a hydrophobic residue of similar size to the highly polar Asn and designed to retain the cis bond, revealed the importance of hydrogen bonding to bound substrate with both collagenolytic and peptidic activities reduced approximately 3-fold. In contrast, the specificity for type I collagen of the mutant Y189F dropped 3-fold without any significant alteration in general peptidase activity. Therefore, S(3)' and in particular the hydrogen bonding potential of Tyr(189) is a specific molecular determinant for MMP-8 triple helicase activity. The cis bond connection to Asn(188) juxtaposes these two side chains for closely spaced hydrogen bonding with substrate that improves collagenolytic and general catalytic efficiency that could be exploited for new collagenase-specific inhibitor drugs.     

Substrate binding mode and its implication on drug design for botulinum neurotoxin A

The seven antigenically distinct serotypes of Clostridium botulinum neurotoxins, the causative agents of botulism, block the neurotransmitter release by specifically cleaving one of the three SNARE proteins and induce flaccid paralysis. The Centers for Disease Control and Prevention (CDC) has declared them as Category A biowarfare agents. The most potent among them, botulinum neurotoxin type A (BoNT/A), cleaves its substrate synaptosome-associated protein of 25 kDa (SNAP-25). An efficient drug for botulism can be developed only with the knowledge of interactions between the substrate and enzyme at the active site. Here, we report the crystal structures of the catalytic domain of BoNT/A with its uncleavable SNAP-25 peptide (197)QRATKM(202) and its variant (197)RRATKM(202) to 1.5 A and 1.6 A, respectively. This is the first time the structure of an uncleavable substrate bound to an active botulinum neurotoxin is reported and it has helped in unequivocally defining S1 to S5' sites. These substrate peptides make interactions with the enzyme predominantly by the residues from 160, 200, 250 and 370 loops. Most notably, the amino nitrogen and carbonyl oxygen of P1 residue (Gln197) chelate the zinc ion and replace the nucleophilic water. The P1'-Arg198, occupies the S1' site formed by Arg363, Thr220, Asp370, Thr215, Ile161, Phe163 and Phe194. The S2' subsite is formed by Arg363, Asn368 and Asp370, while S3' subsite is formed by Tyr251, Leu256, Val258, Tyr366, Phe369 and Asn388. P4'-Lys201 makes hydrogen bond with Gln162. P5'-Met202 binds in the hydrophobic pocket formed by the residues from the 250 and 200 loop. Knowledge of interactions between the enzyme and substrate peptide from these complex structures should form the basis for design of potent inhibitors for this neurotoxin.