Three dimensional structures of S189D chymotrypsin and D189S trypsin mutants: the effect of polarity at site 189 on a protease-specific stabilization of the substrate-binding site

The crystal structure of S189D rat chymotrypsin have been determined (resolution 2.55A) and compared, together with D189S rat trypsin to wild-type structures to examine why these single mutations resulted in poorly active, non-specific enzymes instead of converting the specificities of trypsin and chymotrypsin into each other. Both mutants have stable structure but suffer from a surprisingly large number of serious deformations. These are restricted to the activation domain, mainly to the substrate-binding region and are larger in S189D chymotrypsin. A wild-type substrate-binding mode in the mutants is disfavored by substantial displacements of the Cys191-Cys220 disulfide and loop segments 185-195 (loop C2/D2) and 217-224 (loop E2/F2) at the specificity site. As a consequence, the substrate-binding clefts become wider and more solvent-accessible in the middle third and occluded in the lower third. Interestingly, while the Ser189 residue in D189S trypsin adopts a chymotrypsin-like conformation, the Asp189 residue in S189D chymotrypsin is turned out toward the solvent. The rearrangements in D189S trypsin are at the same sites where trypsin and trypsinogen differ and, in S189D chymotrypsin, the oxyanion hole as well as the salt-bridge between Asp194 and the N-terminal of Ile16 are missing as in chymotrypsinogen. Despite these similarities, the mutants do not have zymogen conformation. The Ser189Asp and Asp189Ser substitutions are structurally so disruptive probably because the stabilization of such a different specificity site polarities as those after the removal or introduction of a charged residue are beyond the capability of the wild-type conformation of the substrate-binding region.     

The protein S-binding site localized to the central core of C4b-binding protein

Human C4b-binding protein (C4BP) is a regulator of the classical pathway of the complement system. It appears in two forms in plasma, as free protein and in a noncovalent complex with the vitamin K-dependent coagulation protein, protein S. In the electron microscope C4BP has a spider-like structure with a central core and seven extended tentacles, each of which has a binding site for C4b, although the protein S-binding site has not been unequivocally pinpointed. C4BP was subjected to chymotrypsin digestion which yielded two major fragments, one of 160 kDa representing the central core, and one of 48 kDa representing the cleaved-off tentacles. We have now localized the protein S-binding site to the 160-kDa central core fragment. Using immunoblotting with a panel of polyclonal antisera, the isolated central core was shown to be completely devoid of 48-kDa fragments. The protein S-binding site was susceptible to proteolysis by chymotrypsin, but was protected by a molar excess of protein S included during the proteolysis. The 160-kDa central core fragment consisted of identical, disulfide-linked 25-kDa peptides and a proper disulfide bond arrangement was crucial to protein S binding. Using a direct binding assay it was shown that the isolated central core had the same affinity for protein S as intact C4BP.     

The crystal structure of recombinant rat pancreatic RNase A

The three-dimensional structure of rat pancreatic RNase A expressed in Escherichia coli was determined. The backbone conformations of certain critical loops are significantly different in this enzyme compared to its bovine counterpart. However, the core structure of rat RNase A is similar to that of the other members of the pancreatic ribonuclease family. The structural variations within a loop bordering the active site can be correlated with the subtle differences in the enzymatic activities of bovine and rat ribonucleases for different substrates. The most significant difference in the backbone conformation was observed in the loop 15-25. This loop incorporates the subtilisin cleavage site which is responsible for RNase A to RNase S conversion in the bovine enzyme. The rat enzyme does not get cleaved under identical conditions. Molecular docking of this region of the rat enzyme in the active site of subtilisin shows steric incompatibility, although the bovine pancreatic ribonuclease A appropriately fits into this active site. It is therefore inferred that the local conformation of the substrate governs the specificity of subtilisin.     

Crystal structure of bovine duodenase, a serine protease, with dual trypsin and chymotrypsin-like specificities

The three-dimensional structure of duodenase, a serine protease from bovine duodenum mucosa, has been determined at 2.4A resolution. The enzyme, which has both trypsin-like and chymotrypsin-like activities, most closely resembles human cathepsin G with which it shares 57% sequence identity and similar specificity. The catalytic Ser195 in duodenase adopts the energetically favored conformation typical of serine proteinases and unlike the strained state typical of lipase/esterases. Of several waters in the active site of duodenase, the one associated with Ser214 is found in all serine proteinases and most lipase/esterases. The conservation of the Ser214 residue in serine proteinase, its presence in the active site, and participation in a hydrogen water network involving the catalytic triad (His57, Asp107, and Ser195) argues for its having an important role in the mechanism of action. It may be referred to as a fourth member of the catalytic triad. Duodenase is one of a growing family of enzymes that possesses trypsin-like and chymotrypsin-like activity. Not long ago, these activities were considered to be mutually exclusive. Computer modeling reveals that the S1 subsite of duodenase has structural features compatible with effective accommodation of P1 residues typical of trypsin (Arg/Lys) and chymotrypsin (Tyr/Phe) substrates. The determination of structural features associated with functional variation in the enzyme family may permit design of enzymes with a specific ratio of trypsin and chymotrypsin activities.     

Structural consequences of accommodation of four non-cognate amino acid residues in the S1 pocket of bovine trypsin and chymotrypsin

Crystal structures of P1 Gly, Val, Leu and Phe bovine pancreatic trypsin inhibitor (BPTI) variants in complex with two serine proteinases, bovine trypsin and chymotrypsin, have been determined. The association constants for the four mutants with the two enzymes show that the enlargement of the volume of the P1 residue is accompanied by an increase of the binding energy, which is more pronounced for bovine chymotrypsin. Since the conformation of the P1 side-chains in the two S1 pockets is very similar, we suggest that the difference in DeltaG values between the enzymes must arise from the more polar environment of the S1 site of trypsin. This results mainly from the substitutions of Met192 and Ser189 observed in chymotrypsin with Gln192 and Asp189 present in trypsin. The more polar interior of the S1 site of trypsin is reflected by a much higher order of the solvent network in the empty pocket of the enzyme, as is observed in the complexes of the two enzymes with the P1 Gly BPTI variant. The more optimal binding of the large hydrophobic P1 residues by chymotrypsin is also reflected by shrinkage of the S1 pocket upon the accommodation of the cognate residues of this enzyme. Conversely, the S1 pocket of trypsin expands upon binding of such side-chains, possibly to avoid interaction with the polar residues of the walls. Further differentiation between the two enzymes is achieved by small differences in the shape of the S1 sites, resulting in an unequal steric hindrance of some of the side-chains, as observed for the gamma-branched P1 Leu variant of BPTI, which is much more favored by bovine chymotrypsin than trypsin. Analysis of the discrimination of beta-branched residues by trypsin and chymotrypsin is based on the complexes with the P1 Val BPTI variant. Steric repulsion of the P1 Val residue by the walls of the S1 pocket of both enzymes prevents the P1 Val side-chain from adopting the most optimal chi1 value.     

Chymotrypsin inhibitory conformation of dipeptides constructed by side chain–side chain hydrophobic interactions

A complete series of configurationally isomers (L -L , L -D , D -L AND D -D ) of a dipeptide Leu-Phe benzyl ester have been synthesized and assayed for chymotrypsin. In the conformational analysis by 400 MMz ¹H NMR, the L -D and D -L isomers, but not hte L -L and D -D isomers, showed fairly large up field shifts (0.2–0.4 ppm) of Leu-βCH₂ and γCH proton signals, indicating the presence of shielding effects from the benzene ring. In addition to distinct signal splitting of Phe-βCH₂, the NOE enhancement observed between Leu-δCH₃ and Phe-phenyl groups revealed that these groups are in close proximity. These data indicated that L -D and D -L isomers from a hydrophobic core between side chains of adjacent Leu and Phe residues. When the dipeptides were examined for inhibition of chymotrypsin using Ac-Try-OEt as a substrate, the L -L isomer showed no inhibition, itself becoming a substrate. However, the other three isomers inhibited chymotrypsin in a competitive manner, and the D -L isomer was strongest with K_i of 2.2 × 10_?5 M . It was found that the D -L isomer was only slowly hydrolysed but the L (or D )-D isomer was not. H-D -Phe-L -Leu-OBzl with the inverse sequence of H-D -Leu-L -Pre-OBzl inhibited chymotrypsin more strongly (K_i = 6.3 × 10^?6 M ). Since the free acid analogue of the D -L isomer exhibited no inhibition, the benzyl ester moiety itself was thought to be involved in the enzyme inhibition. It is assumed that in the inhibitory conformation the ester-benzyl group fits the S₁ site of chymotrypsin, while the side chain-side chain complexing hydrophobic core fits the S₂ site.     

Crystal structure of gamma-chymotrypsin in complex with 7-hydroxycoumarin

The 1.8 A crystal structure of 7-hydroxycoumarin (7-HC) bound to chymotrypsin reveals that this inhibitor forms a planar cinnamate acyl-enzyme complex. The phenyl ring of the bound inhibitor forms numerous van der Waals contacts in the S1 pocket of the enzyme, with the p-hydroxyl group donating a hydrogen bond to the main-chain oxygen atom of Ser217, and the o-hydroxyl group forming a water-mediated hydrogen bond with the carbonyl oxygen of Val227. The structure of the acyl-enzyme complex suggests that the mechanism of inhibition of 7-HC involves nucleophilic attack by the Ser195 O(gamma) atom on the carbonyl carbon atom of the inhibitor, accompanied by the breaking of the 2-pyrone ring of the inhibitor, and leading to the formation of a cinnamate acyl-enzyme derivative via a tetrahedral transition state. Comparisons with structures of photoreversible cinnamates bound to chymotrypsin reveal that although 7-HC interacts with the enzyme in a similar fashion, the binding of 7-HC to chymotrypsin takes place in a productive conformation in contrast to the photoreversible cinnamates. In summary, the 7-HC-chymotrypsin complex provides basic insight into the inhibition of chymotrypsin by natural coumarins and provides a structural basis for the design of more potent mechanism-based inhibitors against a wide range of biologically important chymotrypsin-like enzymes.     

Characterization of the interaction between human protein S and C4b-binding protein (C4bp)

C4b-binding protein, C4bp, is a regulatory factor of the complement system and is also known to be a binding protein of vitamin K-dependent coagulation factor, protein S. Whereas the C4b-binding site is known to be located in the middle part of the subunit chains of C4bp, the location and properties of protein S-binding site are uncertain. Therefore, we have examined the characteristics of the interaction between human protein S and C4bp. Proteolysis of C4bp-protein S complex with chymotrypsin yielded N-terminal-derived 48-kDa fragments of C4bp subunit chains and a C-terminal-derived 160-kDa core fragment of C4bp, to which protein S was still bound. This result suggested that the protein S-binding site is located in the core domain of C4bp. Gel filtration of guanidine-treated C4bp-protein S complex in the absence of guanidine resulted in the separation of C4bp and protein S. Binding assay with 125I-labeled protein S showed that the guanidine-treated C4bp lacked the protein S-binding activity. This result suggests that the protein S-binding site in C4bp is denatured irreversibly by guanidine treatment and therefore seems to be dependent on a specific conformation of C4bp. The C4bp-binding site of protein S was lost upon thrombin treatment, suggesting that the N-terminal thrombin-sensitive region of protein S may be related to the C4bp-binding site. Although free protein S was susceptible to chymotrypsin, leukocyte elastase, and cathepsin G, C4bp-bound protein S was found to be resistant to these proteases.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dissection study on the severe acute respiratory syndrome 3C-like protease reveals the critical role of the extra domain in dimerization of the enzyme: defining the extra domain as a new target for design of highly specific protease inhibitors

The severe acute respiratory syndrome (SARS) 3C-like protease consists of two distinct folds, namely the N-terminal chymotrypsin fold containing the domains I and II hosting the complete catalytic machinery and the C-terminal extra helical domain III unique for the coronavirus 3CL proteases. Previously the functional role of this extra domain has been completely unknown, and it was believed that the coronavirus 3CL proteases share the same enzymatic mechanism with picornavirus 3C proteases, which contain the chymotrypsin fold but have no extra domain. To understand the functional role of the extra domain and to characterize the enzyme-substrate interactions by use of the dynamic light scattering, circular dichroism, and NMR spectroscopy, we 1) dissected the full-length SARS 3CL protease into two distinct folds and subsequently investigated their structural and dimerization properties and 2) studied the structural and binding interactions of three substrate peptides with the entire enzyme and its two dissected folds. The results lead to several findings; 1) although two dissected parts folded into the native-like structures, the chymotrypsin fold only had weak activity as compared with the entire enzyme, and 2) although the chymotrypsin fold remained a monomer within a wide range of protein concentrations, the extra domain existed as a stable dimer even at a very low concentration. This observation strongly indicates that the extra domain contributes to the dimerization of the SARS 3CL protease, thus, switching the enzyme from the inactive form (monomer) to the active form (dimer). This discovery not only separates the coronavirus 3CL protease from the picornavirus 3C protease in terms of the enzymatic mechanism but also defines the dimerization interface on the extra helical domain as a new target for design of the specific protease inhibitors. Furthermore, the determination of the preferred solution conformation of the substrate peptide S1 together with the NMR differential line-broadening and transferred nuclear Overhauser enhancement study allows us to pinpoint the bound structure of the S1 peptide.     

Calpain inhibition by alpha-ketoamide and cyclic hemiacetal inhibitors revealed by X-ray crystallography

Calpains are intracellular calcium-activated cysteine proteases whose unregulated proteolysis following the loss of calcium homeostasis can lead to acute degeneration during ischemic episodes and trauma, as well as Alzheimer's disease and cataract formation. The determination of the crystal structure of the proteolytic core of mu-calpain (muI-II) in a calcium-bound active conformation has made structure-guided design of active site inhibitors feasible. We present here high-resolution crystal structures of rat muI-II complexed with two reversible calpain-specific inhibitors employing cyclic hemiacetal (SNJ-1715) and alpha-ketoamide (SNJ-1945) chemistries that reveal new details about the interactions of inhibitors with this enzyme. The SNJ-1715 complex confirms that the free aldehyde is the reactive species of the cornea-permeable cyclic hemiacetal. The alpha-ketoamide warhead of SNJ-1945 binds with the hydroxyl group of the tetrahedral adduct pointing toward the catalytic histidine rather than the oxyanion hole. The muI-II-SNJ-1945 complex shows residue Glu261 displaced from the S1' site by the inhibitor, resulting in an extended "open" conformation of the domain II gating loop and an unobstructed S1' site. This conformation offers an additional template for structure-based drug design extending to the primed subsites. An important role for the highly conserved Glu261 is proposed.     

On the domain construction of the multienzyme gramicidin S synthetase 2. Isolation of domains activating valine and leucine

The multienzyme gramicidin S synthetase 2, composed of one polypeptide chain, was treated with trypsin and chymotrypsin to give fragments retaining partial enzyme activities. Previously, a tryptic fragment of this multienzyme has been identified as a structural and functional domain. In this study two more fragments, activating Leu and Val, respectively, are shown to represent domains. Careful inspection of the data on limited proteolysis, from this study as well as from previous work, suggests that domains are not simply connected like pearls on a string, and a model for the structure of gramicidin S synthetase, with implications for other peptide synthetase multienzymes, is presented. It is suggested that gramicidin S synthetase 2 is constructed from core catalytic domains and intervening framework. Such an interpretation is in accordance with all published data on limited proteolysis of peptide synthetases, but needs an interplay with gene structural studies in order to be validated and refined.     

Novel subunit in C4b-binding protein required for protein S binding

C4b-binding protein (C4BP) is a multimeric protein with regulatory functions in the complement system. It also interacts with vitamin K-dependent protein S, which is involved in the regulation of the coagulation system. It has been demonstrated that C4BP consists of seven disulfide-linked, identical 70-kDa subunits, which are arranged to give the molecule a spider-like structure. We now have evidence for the presence of a new subunit in C4BP. On sodium dodecyl sulfate-poly-acrylamide gel electrophoresis it appears as a weakly stainable band with a molecular weight of approximately 45,000. The subunit was isolated by gel filtration in 6 M guanidine hydrochloride of reduced and carboxymethylated C4BP. Its amino-terminal sequence is distinct from previously known protein sequences. The stoichiometry of 45- to 70-kDa subunits was estimated to be 1:9, indicating the presence of one 45-kDa subunit per C4BP molecule. The new subunit was demonstrated to be a disulfide-linked component of the central core of C4BP. It was sensitive to proteolysis by chymotrypsin, and when cleaved the protein S binding ability of C4BP was lost. With protein S bound to C4BP, the 45-kDa subunit was protected from degradation by chymotrypsin, and the protein S binding site remained intact. These data suggest that the new subunit is directly involved in protein S binding.     

Structure and function studies of the cGMP-stimulated phosphodiesterase.

Studies of cGMP binding to both the native cyclic GMP-stimulated phosphodiesterase and to two unique isolated chymotryptic fragments lacking the catalytic domain suggest that the enzyme contains two noncatalytic cGMP-binding sites/homodimer. In the presence of high concentrations of ammonium sulfate, 2 mol of cGMP are bound/mol of cGMP-stimulated phosphodiesterase homodimer. Under these conditions, linear Scatchard plots of binding are obtained that give an apparent Kd of approximately 2 microM. The inclusion of 3-isobutyl-1-methylxanthine produces a curvilinear plot. In the absence of ammonium sulfate, the dissociation of cGMP from the holoenzyme is rapid, having a t1/2 of less than 10 s, and addition of ammonium sulfate to the incubation greatly decreases this rate of dissociation. The native enzyme is resistant to degradation by chymotrypsin in the absence of cGMP; however, in its presence, chymotrypsin treatment produces several discrete fragments. Similarly, in the presence but not in the absence of cGMP, dicyclohexylcarbodiimide causes an irreversible activation of the enzyme without cross-linking the nucleotide to the phosphodiesterase. Both observations provide evidence that a different conformation in the enzyme results from cGMP binding. Only the conformation formed upon cGMP binding is easily attacked by chymotrypsin or permanently activated by treatment with dicyclohexylcarbodiimide. One major chymotryptic cleavage site exposed by cGMP binding is at tyrosine 553, implying that this region takes part in the conformational change. Limited proteolysis experiments indicate that these noncatalytic binding sites are located within a region of internal sequence homology previously proposed to include the cGMP-binding site(s) and that they retain a high affinity and specificity for cGMP independent of the catalytic domain of the enzyme. The products formed by partial proteolysis can be separated into individual catalytically active and cGMP-binding fractions by anion exchange chromatography. Gel filtration and electrophoresis analysis of the isolated fractions suggest that the cGMP-binding peak has a dimeric structure. Moreover, it can be further resolved by polyethyleneimine high performance liquid chromatography into two peaks (Peaks IIIA and IIIB). Peak IIIA binds 2 mol of cGMP/mol of dimer with an apparent Kd of 0.2 microM. Peak IIIB, however, has greatly reduced cGMP binding. Further digestion of these fragments with cyanogen bromide show that the differences between Peaks IIIA and IIIB are due to one or more additional proteolytic nicks in IIIB that remove a few residues near its C terminus, most probably residues 523-550 or 534-550. This in turn suggests that this region is essential for cGMP-binding activity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Intramolecular interactions of the regulatory region with the catalytic core in the plasma membrane calcium pump

The access of three proteases to their sites of cleavage was used as a measure of regulatory interactions in the plasma membrane Ca2+ pump isoform 4b (PMCA4b). When the proteases could not cut at their sites in the C-terminal regulatory region, the interaction was judged to be tight. This was the case in the absence of Ca2+, when chymotrypsin and caspase cut PMCA only very slowly. Ca2+ accelerated the fragmentation, but the digestion remained incomplete. In the presence of Ca2+ plus calmodulin, the digestion became nearly complete in all cases, indicating a more flexible conformation of the carboxyl terminus in the fully activated state. The acceleration of proteolysis by Ca2+ or Ca2+ plus calmodulin occurred equally at the caspase site upstream of the calmodulin-binding domain and the chymotrypsin and calpain sites downstream of that domain. Replacing Trp1093 (a key residue within the calmodulin-binding domain) with alanine had a much more specific effect, because it exposed only proteolytic sites within the calmodulin-binding domain that had previously been shielded in the native protein. At these sites, both calpain and chymotrypsin cut the Trp1093 --> Ala mutant in the absence of calmodulin. These data indicate that, in the auto-inhibited conformation, the calmodulin-binding/auto-inhibitory sequence and the regions both upstream and downstream are in close contact with the catalytic core. Trp1093 plays an essential role not only in stabilizing the Ca2+-calmodulin/calmodulin-binding domain complex but also in the formation or stability of the inhibitory conformation of that domain when it interacts with the catalytic core of PMCA4b.     

A spin label study of immobilized enzyme spectral subpopulations

Electron spin resonance (ESR) spin label studies have been carried out to examine the active site conformation of alpha-chymotrypsin before and after immobilization on two types of organic polymer supports: Amberlite XAD-8 and XAD-2. alpha-Chymotryspin was first chemically modified by reaction with methyl-4-phenylbutyrimidate and then inhibited by the active site spin label 4-(2,2,6,6-tetramethyl-piperdine-1-oxyl)-m-flurosulfonylbenzamide. In general, the ESR spectra of the active site lable revealed no significant changes in conformation for most of the enzyme before or after derivatization. On the other hand, two spectral subpopulations (A and B) of spin-labeled enzyme were characterized on the basis of their ESR spectra after immobilization on Amberlite XAD-8. Spectral subpopulation A (distinguished by a highly restrained spectrum) appeared to retain its active site structure and conformation and represented a large majority of the labeled chymotrypsin on the beads. Its presence correlated with the high activity and stability of phenylbutyramidinated chymotryspin on the Amberlite XAD-8 beads. Spectral subpopulation B (distinguished by a very weakly constrained spectrum) appeared to reflect loosely bound or denatured enzyme which was removable upon washing with 40% (v/v) ethylene glycol. Two methods for examining solvent accessibility to the active site lable of the kinetics of ascorbate reduction suggested that both spectral subpopulations had identical accessibilities to the bulk solvent. Paramagnetic broadening of the signal by K(3)Fe(CN)(6) revealed differences in the spin-spin broadening of the A and B components but is deemed and inappropriate indicator of solvent accessibility.     

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.     

The reaction of human alpha 2-macroglobulin with alpha-chymotrypsin. A stopped-flow kinetic investigation

The kinetics of the conformational changes of human alpha 2-macroglobulin (alpha 2M) induced by reaction with pure alpha-chymotrypsin, have been analyzed using three fluorescent probes, namely protein tryptophan groups and the dye 6-(4-toluidino)-2-naphthalenesulfonate, to monitor alterations of the alpha 2M structure, and a covalent conjugate of chymotrypsin and fluorescein isothiocyanate (Chy-FITC). The main reaction sequence exhibits a triphasic time course with any of the labels used. Each phase is first-order. The fixation of a single molecule of chymotrypsin to one protease-binding site of alpha 2M (site A) initiates the whole process and determines the access to the second site (site B). Of the three exponential phases of the reaction (20 degrees C), phase I (k1 approximately 19.6 min-1) and phase II (k2 approximately 5.3 min-1) belong to site A. Phase III is related to site B transformation. It contains two steps with different responses from tryptophan (k3 approximately 0.77 min-1) and Chy-FITC (k3 approximately 0.19 min-1) fluorescence measurements. The point to be stressed is that site A and site B, while presumably identical in the native form, are not equivalent with regard to their fluorescence and kinetic properties. However, the activation energy (E = 30.1 +/- 2.7 kJ mol-1) is the same for the three phases of the reaction. When present in sufficient excess, free chymotrypsin or native alpha 2M is able to form reversible complexes with the above-related chymotrypsin-alpha 2M adducts. Only the alpha 2M site A core seems to be involved in this parallel process. In addition the conformational state of the chymotrypsin-alpha 2M complexes is shown to depend on the pH, with a pKa of 6.4.     

NMR solution structure of Apis mellifera chymotrypsin/cathepsin G inhibitor-1 (AMCI-1): structural similarity with Ascaris protease inhibitors

The three-dimensional structure of the 56 residue polypeptide Apis mellifera chymotrypsin/cathepsin G inhibitor 1 (AMCI-1) isolated from honey bee hemolymph was calculated based on 730 experimental NMR restraints. It consists of two approximately perpendicular beta-sheets, several turns, and a long exposed loop that includes the protease binding site. The lack of extensive secondary structure features or hydrophobic core is compensated by the presence of five disulfide bridges that stabilize both the protein scaffold and the binding loop segment. A detailed analysis of the protease binding loop conformation reveals that it is similar to those found in other canonical serine protease inhibitors. The AMCI-1 structure exhibits a common fold with a novel family of inhibitors from the intestinal parasitic worm Ascaris suum. The pH-induced conformational changes in the binding loop region observed in the Ascaris inhibitor ATI are absent in AMCI-1. Similar binding site sequences and structures strongly suggest that the lack of the conformational change can be attributed to a Glu-->Gln substitution at the P1' position in AMCI-1, compared to ATI. Analysis of amide proton temperature coefficients shows very good correlation with the presence of hydrogen bond donors in the calculated AMCI-1 structure.     

Properties of p-hydroxybenzoate hydroxylase when stabilized in its open conformation

p-Hydroxybenzoate hydroxylase is extensively studied as a model for single-component flavoprotein monooxygenases. It catalyzes a reaction in two parts: (1) reduction of the FAD in the enzyme by NADPH in response to binding of p-hydroxybenzoate to the enzyme and (2) oxidation of reduced FAD with oxygen in an environment free from solvent to form a hydroperoxide, which then reacts with p-hydroxybenzoate to form an oxygenated product. These different reactions are coordinated through conformational rearrangements of the protein and the isoalloxazine ring during catalysis. Until recently, it has not been clear how p-hydroxybenzoate gains access to the buried active site. In 2002, a structure of a mutant form of the enzyme without substrate was published that showed an open conformation with solvent access to the active site [Wang, J., Ortiz-Maldonado, M., Entsch, B., Massey, V., Ballou, D., and Gatti, D. L. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 608-613]. The wild-type enzyme does not form high-resolution crystals without substrate. We hypothesized that the wild-type enzyme without substrate also forms an open conformation for binding p-hydroxybenzoate, but only transiently. To test this idea, we have studied the properties of two different mutant forms of the enzyme that are stabilized in the open conformation. These mutant enzymes bind p-hydroxybenzoate very fast, but with very low affinity, as expected from the open structure. The mutant enzymes are extremely inactive, but are capable of slowly forming small amounts of product by the normal catalytic pathway. The lack of activity results from the failure of the mutants to readily form the out conformation required for flavin reduction by NADPH. The mutants form a large fraction of an abnormal conformation of the reduced enzyme with p-hydroxybenzoate bound. This conformation of the enzyme is unreactive with oxygen. We conclude that transient formation of this open conformation is the mechanism for sequestering p-hydroxybenzoate to initiate catalysis. This overall study emphasizes the role that protein dynamics can play in enzymatic catalysis.     

Crystallographic evidence for substrate ring distortion and protein conformational changes during catalysis in cellobiohydrolase Ce16A from trichoderma reesei.

BACKGROUND: Cel6A is one of the two cellobiohydrolases produced by Trichoderma reesei. The catalytic core has a structure that is a variation of the classic TIM barrel. The active site is located inside a tunnel, the roof of which is formed mainly by a pair of loops. RESULTS: We describe three new ligand complexes. One is the structure of the wild-type enzyme in complex with a nonhydrolysable cello-oligosaccharide, methyl 4-S-beta-cellobiosyl-4-thio-beta-cellobioside (Glc)(2)-S-(Glc)(2), which differs from a cellotetraose in the nature of the central glycosidic linkage where a sulphur atom replaces an oxygen atom. The second structure is a mutant, Y169F, in complex with the same ligand, and the third is the wild-type enzyme in complex with m-iodobenzyl beta-D-glucopyranosyl-beta(1,4)-D-xylopyranoside (IBXG). CONCLUSIONS: The (Glc)(2)-S-(Glc)(2) ligand binds in the -2 to +2 sites in both the wild-type and mutant enzymes. The glucosyl unit in the -1 site is distorted from the usual chair conformation in both structures. The IBXG ligand binds in the -2 to +1 sites, with the xylosyl unit in the -1 site where it adopts the energetically favourable chair conformation. The -1 site glucosyl of the (Glc)(2)-S-(Glc)(2) ligand is unable to take on this conformation because of steric clashes with the protein. The crystallographic results show that one of the tunnel-forming loops in Cel6A is sensitive to modifications at the active site, and is able to take on a number of different conformations. One of the conformational changes disrupts a set of interactions at the active site that we propose is an integral part of the reaction mechanism.