Procarboxypeptidase A from the insect pest Helicoverpa armigera and its derived enzyme. Two forms with new functional properties.

Although there is a significant knowledge about mammalian metallocarboxypeptidases, the data available on this family of enzymes is very poor for invertebrate forms. Here we present the biochemical characterization of a metallocarboxypeptidase from the insect Helicoverpa armigera (Lepidoptera: Noctuidae), a devastating pest spread in subtropical regions of Europe, Asia, Africa and Oceania. The zymogen of this carboxypeptidase (PCPAHa) has been expressed at high levels in a Pichia pastoris system and shown to display the characteristics of the enzyme purified from the insect midgut. The in vitro activation process of the proenzyme differs significantly from the mammalian ones. The lysine-specific endoprotease LysC activates PCPAHa four times more efficiently than trypsin, the general activating enzyme for all previously studied metalloprocarboxypeptidases. LysC and trypsin independently use two different activation targets and the presence of sugars in the vicinity of the LysC activation point affects the activation process, indicating a possible modulation of the activation mechanism. During the activation with LysC the prodomain is degraded, while the carboxypeptidase moiety remains intact except for a C-terminal octapeptide that is rapidly released. Interestingly, the sequence at the cleavage point for the release of the octapeptide is also found at the boundary between the activation peptide and the enzyme moieties. The active enzyme (CPAHa) is shown to have a very broad substrate specificity, as it appears to be the only known metallocarboxypeptidase capable of efficiently hydrolysing basic and aliphatic residues and, to a much lower extent, acidic residues. Two carboxypeptidase inhibitors, from potato and leech, were tested against CPAHa. The former, of vegetal origin, is the most efficient metallocarboxypeptidase inhibitor described so far, with a Ki in the pm range.     

Rat mast cell carboxypeptidase: amino acid sequence and evidence of enzyme activity within mast cell granules

The amino acid sequence of rat mast cell carboxypeptidase has been determined. The major form has 308 residues; a minor form has an additional (glutamyl) residue at the amino terminus that may indicate an alternate cleavage site during zymogen activation. The enzyme is homologous to pancreatic carboxypeptidases A and B, with conservation of the functional amino acid residues of the active site. The putative substrate binding site resembles that of carboxypeptidase A, although other structural features bear more similarity to carboxypeptidase B. Mast cell carboxypeptidase retains enzymatic activity toward a peptide substrate (angiotensin I) while bound within the granular matrix of the rat connective tissue mast cells. Evidence is presented to suggest that a cluster of positively charged lysyl and arginyl residues binds the enzyme to the negatively charged heparin of the granular matrix but leaves the active site exposed to bind and cleave peptide substrates.     

Identification of a novel prohormone sorting signal-binding site on carboxypeptidase E, a regulated secretory pathway-sorting receptor

Sorting of the prohormone POMC to the regulated secretory pathway necessitates the binding of a sorting signal to a sorting receptor, identified as membrane carboxypeptidase E (CPE). The sorting signal, located at the N terminus of POMC consists of two acidic (Asp10, Glu14) and two hydrophobic (Leu11, Leu18) residues exposed on the surface of an amphipathic loop. In this study, molecular modeling of CPE predicted that the acidic residues in the POMC-sorting signal bind specifically to two basic residues, Arg255 and Lys260, present in a loop unique to CPE, compared with other carboxypeptidases. To test the model, these two residues on CPE were mutated to Ser or Ala, followed by baculovirus expression of the mutant CPEs in Sf9 cells. Sf9 cell membranes containing CPE mutants with either Arg255 or Lys260, or both residues substituted, showed no binding of [125I]N-POMC1-26 (which contains the POMC-sorting signal motif), proinsulin, or proenkephalin. In contrast, substitution of an Arg147 to Ala147 at a substrate-binding site, Arg259 to Ala259 and Ser202 to Pro202, in CPE did not affect the level of [125I]N-POMC1-26 binding when compared with-wild type CPE. Furthermore, mutation of the POMC-sorting signal motif (Asp10, Leu11, Glu14, Leu18) eliminated binding to wild-type CPE. These results indicate that the sorting signal of POMC, proinsulin, and proenkephalin specifically interacts with Arg255 and Lys260 at a novel binding site, independent of the active site on CPE.     

Characterization of a digestive carboxypeptidase from the insect pest corn earworm (Helicoverpa armigera) with novel specificity towards C-terminal glutamate residues.

Carboxypeptidases were purified from guts of larvae of corn earworm (Helicoverpa armigera), a lepidopteran crop pest, by affinity chromatography on immobilized potato carboxypeptidase inhibitor, and characterized by N-terminal sequencing. A larval gut cDNA library was screened using probes based on these protein sequences. cDNA HaCA42 encoded a carboxypeptidase with sequence similarity to enzymes of clan MC [Barrett, A. J., Rawlings, N. D. & Woessner, J. F. (1998) Handbook of Proteolytic Enzymes. Academic Press, London.], but with a novel predicted specificity towards C-terminal acidic residues. This carboxypeptidase was expressed as a recombinant proprotein in the yeast Pichia pastoris. The expressed protein could be activated by treatment with bovine trypsin; degradation of bound pro-region, rather than cleavage of pro-region from mature protein, was the rate-limiting step in activation. Activated HaCA42 carboxypeptidase hydrolysed a synthetic substrate for glutamate carboxypeptidases (FAEE, C-terminal Glu), but did not hydrolyse substrates for carboxypeptidase A or B (FAPP or FAAK, C-terminal Phe or Lys) or methotrexate, cleaved by clan MH glutamate carboxypeptidases. The enzyme was highly specific for C-terminal glutamate in peptide substrates, with slow hydrolysis of C-terminal aspartate also observed. Glutamate carboxypeptidase activity was present in larval gut extract from H. armigera. The HaCA42 protein is the first glutamate-specific metallocarboxypeptidase from clan MC to be identified and characterized. The genome of Drosophila melanogaster contains genes encoding enzymes with similar sequences and predicted specificity, and a cDNA encoding a similar enzyme has been isolated from gut tissue in tsetse fly. We suggest that digestive carboxypeptidases with sequence similarity to the classical mammalian enzymes, but with specificity towards C-terminal glutamate, are widely distributed in insects.     

Crystal structure of the cytochrome P-450CAM active site mutant Thr252Ala.

The crystal structure of a cytochrome P-450CAM site-directed mutant in which the active site Thr252 has been replaced with an Ala (Thr252Ala) has been refined to an R factor of 0.18 at 2.2 A. According to sequence alignments (Nelson & Strobel, 1989), Thr252 is highly conserved among P-450 enzymes. The crystallographic structure of ferrous camphor- and carbon monoxide-bound P-450CAM (Raag & Poulos, 1989b) suggests that Thr252 is a key active site residue, forming part of the dioxygen-binding site. Mutation of the active site threonine to alanine produces an enzyme in which substrate hydroxylation is uncoupled from electron transfer. Specifically, hydrogen peroxide and "excess" water are produced instead of the product, 5-exo-hydroxycamphor. The X-ray structure has revealed that a local distortion in the distal helix between Gly248 and Thr252 becomes even more severe in the Thr252Ala mutant. Furthermore, a solvent molecule not present in the native enzyme is positioned in the dioxygen-binding region of the mutant enzyme active site. In this location, the solvent molecule could sterically interfere with and destabilize dioxygen binding. In addition, the active site solvent molecule is connected, via a network of hydrogen bonds, with an internal solvent channel which links distal helix residues to a buried Glu side chain. Thus, solvent protons appear to be much more accessible to dioxygen in the mutant than in the wild-type enzyme, a factor which may promote hydrogen peroxide and/or water production instead of substrate hydroxylation. On the basis of crystallographic and mutagenesis data, a proton delivery pathway involving residues Lys178/Arg186, Asp251, and Thr252 is proposed for wild-type P-450CAM. Coordinates of structures discussed in this paper have been submitted to the Brookhaven Protein Data Bank (Bernstein et al., 1977).     

Three-dimensional structure of porcine procarboxypeptidase B: a structural basis of its inactivity.

Procarboxypeptidase B is converted to enzymatically active carboxypeptidase B by limited proteolysis catalysed by trypsin, removing the long N-terminal activation segment of 95 amino acids. The three-dimensional crystal structure of procarboxypeptidase B from porcine pancreas has been determined at 2.3 A resolution and refined to a crystallographic R-factor of 0.169. The functional determinants of its enzymatic inactivity and of its activation by limited proteolysis have thus been unveiled. The activation segment folds in a globular region with an open sandwich antiparallel-alpha antiparallel-beta topology and in a C terminal alpha-helix which connects it to the enzyme moiety. The globular region (A7-A82) shields the preformed active site, and establishes specific interactions with residues important for substrate recognition. AspA41 forms a salt bridge with Arg145, which in active carboxypeptidase binds the C-terminal carboxyl group of substrate molecules. The connecting region occupies the putative extended substrate binding site. The scissile peptide bond cleaved by trypsin during activation is very exposed. Its cleavage leads to the release of the activation segment and to exposure of the substrate binding site. An open-sandwich folding has been observed in a number of other proteins and protein domains. One of them is the C-terminal fragment of L7/L12, a ribosomal protein from Escherichia coli that displays a topology similar to the activation domain of procarboxypeptidase.     

Three subunits contribute amino acids to the active site of tetrameric adenylosuccinate lyase: Lys268 and Glu275 are required

Tetrameric adenylosuccinate lyase (ASL) of Bacillus subtilis catalyzes the cleavage of adenylosuccinate to form AMP and fumarate. We previously reported that two distinct subunits contribute residues to each active site, including the His68 and His89 from one and His141 from a second subunit [Brosius, J. L., and Colman, R. F. (2000) Biochemistry 39, 13336-13343]. Glu(275) is 2.8 A from His141 in the ASL crystal structure, and Lys268 is also in the active site region; Glu275 and Lys268 come from a third, distinct subunit. Using site-directed mutagenesis, we have replaced Lys268 by Arg, Gln, Glu, and Ala, with specific activities of the purified mutant enzymes being 0.055, 0.00069, 0.00028, and 0.0, respectively, compared to 1.56 units/mg for wild-type (WT) enzyme. Glu275 was substituted by Gln, Asp, Ala, and Arg; none of these homogeneous mutant enzymes has detectable activity. Circular dichroism and light scattering reveal that neither the secondary structure nor the oligomeric state of the Lys268 mutant enzymes has been perturbed. Native gel electrophoresis and circular dichroism indicate that the Glu275 mutant enzymes are tetramers, but their conformation is altered slightly. For K268R, the K(m)s for all substrates are similar to WT enzyme. Binding studies using [2-3H]-adenylosuccinate reveal that none of the Glu275 mutant enzymes, nor inactive K268A, can bind substrate. We propose that Lys268 participates in binding substrate and that Glu275 is essential for catalysis because of its interaction with His141. Incubation of H89Q with K268Q or E275Q leads to restoration of up to 16% WT activity, while incubation of H141Q with K268Q or E275Q results in 6% WT activity. These complementation studies provide the first functional evidence that a third subunit contributes residues to each intersubunit active site of ASL. Thus, adenylosuccinate lyase has four active sites per enzyme tetramer, each of which is formed from regions of three subunits.     

The three-dimensional structure of human procarboxypeptidase A2. Deciphering the basis of the inhibition, activation and intrinsic activity of the zymogen.

The three-dimensional structure of human procarboxypeptidase A2 has been determined using X-ray crystallography at 1.8 A resolution. This is the first detailed structural report of a human pancreatic carboxypeptidase and of its zymogen. Human procarboxypeptidase A2 is formed by a pro-segment of 96 residues, which inhibits the enzyme, and a carboxypeptidase moiety of 305 residues. The pro-enzyme maintains the general fold when compared with other non-human counterparts. The globular part of the pro-segment docks into the enzyme moiety and shields the S2-S4 substrate binding sites, promoting inhibition. Interestingly, important differences are found in the pro-segment which allow the identification of the structural determinants of the diverse activation behaviours of procarboxypeptidases A1, B and A2, particularly of the latter. The benzylsuccinic inhibitor is able to diffuse into the active site of procarboxypeptidase A2 in the crystals. The structure of the zymogen-inhibitor complex has been solved at 2.2 A resolution. The inhibitor enters the active site through a channel formed at the interface between the pro-segment and the enzyme regions and interacts with important elements of the active site. The derived structural features explain the intrinsic activity of A1/A2 pro-enzymes for small substrates.     

Structures of two kinetic intermediates reveal species specificity of penicillin-binding proteins

Penicillin-binding proteins (PBPs), the target enzymes of beta-lactam antibiotics such as penicillins and cephalosporins, catalyze the final peptidoglycan cross-linking step of bacterial cell-wall biosynthesis. beta-Lactams inhibit this reaction because they mimic the D-alanyl-D-alanine peptide precursors of cell-wall structure. Prior crystallographic studies have described the site of beta-lactam binding and inhibition, but they have failed to show the binding of D-Ala-D-Ala substrates. We present here the first high-resolution crystallographic structures of a PBP, D-Ala-D-Ala-peptidase of Streptomyces sp. strain R61, non-covalently complexed with a highly specific fragment (glycyl-L-alpha-amino-epsilon-pimelyl-D-Ala-D-Ala) of the cell-wall precursor in both enzyme-substrate and enzyme-product forms. The 1.9A resolution structure of the enzyme-substrate Henri-Michaelis complex was achieved by using inactivated enzyme, which was formed by cross-linking two catalytically important residues Tyr159 and Lys65. The second structure at 1.25A resolution of the uncross-linked, active form of the DD-peptidase shows the non-covalent binding of the two products of the carboxypeptidase reaction. The well-defined substrate-binding site in the two crystallographic structures shows a subsite that is complementary to a portion of the natural cell-wall substrate that varies among bacterial species. In addition, the structures show the displacement of 11 water molecules from the active site, the location of residues responsible for substrate binding, and clearly demonstrate the necessity of Lys65 and or Tyr159 for the acylation step with the donor peptide. Comparison of the complexed structures described here with the structures of other known PBPs suggests the design of species-targeted antibiotics as a counter-strategy towards beta-lactamase-elicited bacterial resistance.     

Interaction of arylsulfatase-A (ASA) with its natural sulfoglycolipid substrates: a computational and site-directed mutagenesis study

Arylsulfatase A (ASA) hydrolyzes sulfate esters with a pH optimum of 5. Interactions between p-nitrocatechol sulfate (NCS, artificial substrate) and active site residues of ASA are revealed from their co-crystal structure. Since equivalent ASA interactions with its natural substrates, sulfogalactosylceramide (SGC) and sulfogalactosylglycerolipid (SGG), are not known, we computationally docked SGC/SGG to the ASA crystal structure. Our dockings suggested that Cys69 was the active site residue, and Lys302 & Lys123 as residues anchoring the sulfate group of SGC/SGG to the active site, as observed for NCS. We further confirmed these results using 2 recombinant ASA mutants: C69A and CKK (Cys69, Lys302 and Lys123-all mutated to Ala). Both ASA mutants failed to desulfate SGC/SGG, and CKK showed minimal binding to [¹⁴C]SGC, although C69A still had affinity for this sulfoglycolipid. However, our dockings suggested additional intermolecular hydrogen bonding and hydrophobic interactions between ASA and SGC/SGG, thus contributing to the specificity of SGC/SGG as natural substrates.     

Tissue factor residues 157-167 are required for efficient proteolytic activation of factor X and factor VII.

The cell surface receptor tissue factor (TF) initiates coagulation by supporting the proteolytic activation of factors X and IX as well as VII to active serine proteases. Architectural similarity of TF to the cytokine receptor family suggests a strand-loop-strand structure for TF residues 151-174. Site-directed Ala exchanges in the predicted surface loop demonstrated that residues Tyr157, Lys159, Ser163, Gly164, Lys165, and Lys166 are important for function. Addition of side chain atoms at the Ser162 position decreased function, whereas the Ala exchange was tolerated. The dysfunctional mutants bound VII with high affinity and fully supported the catalysis of small peptidyl substrates by the mutant TF.VIIa complex. Lys159-->Ala substitution was compatible with efficient activation of factor X, whereas the Try157-->Ala exchange and mutations in the carboxyl aspect of the predicted loop resulted in diminished activation of factor X. The specific plasma procoagulant activity of all functionally deficient mutants increased 7- to 200-fold upon the supplementation of VIIa suggesting that TF residues 157-167 also provide important interactions that accelerate the activation of VII to VIIa. These data are consistent with assignment of the TF 157-167 region as contributing to protein substrate recognition and cleavage by the TF.VIIa complex.     

Structure of a Michaelis complex analogue: propionate binds in the substrate carboxylate site of alanine racemase

The structure of alanine racemase from Bacillus stearothermophilus with the inhibitor propionate bound in the active site was determined by X-ray crystallography to a resolution of 1.9 A. The enzyme is a homodimer in solution and crystallizes with a dimer in the asymmetric unit. Both active sites contain a pyridoxal 5'-phosphate (PLP) molecule in aldimine linkage to Lys39 as a protonated Schiff base, and the pH-independence of UV-visible absorption spectra suggests that the protonated PLP-Lys39 Schiff base is the reactive form of the enzyme. The carboxylate group of propionate bound in the active site makes numerous interactions with active-site residues, defining the substrate binding site of the enzyme. The propionate-bound structure therefore approximates features of the Michaelis complex formed between alanine racemase and its amino acid substrate. The structure also provides evidence for the existence of a carbamate formed on the side-chain amino group of Lys129, stabilized by interactions with one of the residues interacting with the carboxylate group of propionate, Arg136. We propose that this novel interaction influences both substrate binding and catalysis by precisely positioning Arg136 and modulating its charge.     

Contribution of active site residues to the activity and thermal stability of ribonuclease Sa

We have used site-specific mutagenesis to study the contribution of Glu 74 and the active site residues Gln 38, Glu 41, Glu 54, Arg 65, and His 85 to the catalytic activity and thermal stability of ribonuclease Sa. The activity of Gln38Ala is lowered by one order of magnitude, which confirms the involvement of this residue in substrate binding. In contrast, Glu41Lys had no effect on the ribonuclease Sa activity. This is surprising, because the hydrogen bond between the guanosine N1 atom and the side chain of Glu 41 is thought to be important for the guanine specificity in related ribonucleases. The activities of Glu54Gln and Arg65Ala are both lowered about 1000-fold, and His85Gln is totally inactive, confirming the importance of these residues to the catalytic function of ribonuclease Sa. In Glu74Lys, k(cat) is reduced sixfold despite the fact that Glu 74 is over 15 A from the active site. The pH dependence of k(cat)/K(M) is very similar for Glu74Lys and wild-type RNase Sa, suggesting that this is not due to a change in the pK values of the groups involved in catalysis. Compared to wild-type RNase Sa, the stabilities of Gln38Ala and Glu74Lys are increased, the stabilities of Glu41Lys, Glu54Gln, and Arg65Ala are decreased and the stability of His85Gln is unchanged. Thus, the active site residues in the ribonuclease Sa make different contributions to the stability.     

Glu300 of rat carboxypeptidase E is essential for enzymatic activity but not substrate binding or routing to the regulated secretory pathway

Several recently discovered members of the carboxypeptidase E (CPE) gene family lack critical active site residues that are conserved in other family members. For example, three CPE-like proteins contain a Tyr in place of Glu300 (equivalent to Glu270 of carboxypeptidase A and B). To investigate the importance of this position, Glu300 of rat CPE was converted into Gln, Lys, or Tyr, and the proteins expressed in Sf9 cells using the baculovirus system. All three mutants were secreted from the cells, but the media showed no enzyme activity above background levels. Wild-type CPE and the Gln300 point mutant bound to a p-aminobenzoyl-Arg-Sepharose affinity resin, and this binding was competed by an active site-directed inhibitor, guanidinoethylmercaptosuccinic acid. The affinity purified mutant CPE protein showed no detectable enzyme activity (<0.004% of wild-type CPE) toward dansyl-Phe-Ala-Arg. Expression of the Gln300 and Lys300 mutant CPE proteins in the NIT3 mouse pancreatic beta-cell line showed that these mutants are routed into secretory vesicles and secreted via the regulated pathway. Taken together, these results indicate that Glu300 of CPE is essential for enzyme activity, but not required for substrate binding or for routing into the regulated secretory pathway.     

Glycine insertion at protease cleavage site of SNAP25 resists cleavage but enhances affinity for botulinum neurotoxin serotype A

The light chain of botulinum neurotoxin A (BoNT/A‐LC) is a Zn‐dependent protease that specifically cleaves SNAP25 of the SNARE complex, thereby impairing vesicle fusion and neurotransmitter release at neuromuscular junctions. The C‐terminus of SNAP25 (residues 141–206) retains full activity for BoNT/A‐LC‐catalyzed cleavage at P1‐P1' (Gln197‐Arg198). Using the structure of a complex between the C‐terminus of SNAP25 and BoNT/A‐LC as a model to design SNAP25‐derived pseudosubstrate inhibitors (SNAPIs) that prevent presentation of the scissile bond to the active site, we introduced multiple His residues to replace Ala‐Asn‐Gln‐Arg (residues 195–198) at the substrate cleavage site, with the intent to identify possible side‐chain interactions with the active site Zn. We also introduced multiple Gly residues between the P1‐P1' residues to explore the spatial tolerance within the active‐site cleft. Using a FRET substrate YsCsY, we compared a series of SNAPIs for inhibition of BoNT/A‐LC. Among the SNAPIs tested, several known cleavage‐resistant, single‐point mutants of SNAP25 were poor inhibitors, with most of the mutants losing binding affinity. Replacement with His at the active site did not improve inhibition over wildtype substrate. In contrast, Gly‐insertion mutants were not only resistant to cleavage, but also surprisingly showed enhanced affinity for BoNT/A‐LC. Two of the Gly‐insertion mutants exhibited 10‐fold lower IC₅₀ values than the wildtype 66‐mer SNAP25 peptide. Our findings illustrate a scenario, where the induced fit between enzyme and bound pseudosubstrate fails to produce the strain and distortion required for catalysis to proceed.     

Mapping the substrate-binding site of a human class mu glutathione transferase using nuclear magnetic resonance spectroscopy.

The substrate-binding site of a human muscle class mu glutathione transferase has been characterized using high-resolution nuclear magnetic resonance spectroscopy. Isotopic labeling has been used to simplify one-dimensional proton NMR spectra of the Tyr and His residues in the enzyme and two-dimensional carbon-proton spectra of the Ala and Met residues in the enzyme. The resonance lines from 8 of the 12 Tyr residues have been assigned using site-directed mutagenesis. Replacement of Tyr7 with Phe reduced the activity of the enzyme 100-fold. The proximity of His, Tyr, Ala, and Met residues to the active site has been determined using a nitroxide-labeled substrate analogue. This substrate analogue binds with high affinity (Keq = 10(6) M-1) to the enzyme and is a competitive inhibitor. None of the His residues are within 17 A of the active site. Three of the assigned Tyr residues are greater than 17 A from the active site. Quantitative measurement of paramagnetic line broadening of five additional Tyr residues places them within 13-17 A from the active site. Broadening of the Ala and Met resonance lines by the spin-labeled substrate indicates that three Ala residues are 9-16 A from the nitroxide, three Met residues are less than 9 A from the nitroxide, and two Met residues are 9-16 A from the nitroxide.(ABSTRACT TRUNCATED AT 250 WORDS)     

The PD...(D/E)XK motif in restriction enzymes: a link between function and conformation

The active sites of Mg(II)-dependent nucleases feature a cluster of conserved charged residues which includes both acidic (Asp and Glu) and basic (Lys) side chains. In restriction enzymes, these side chains are part of the conserved PD...(D/E)XK functional sequence motif which has been implicated as being important in metal ion binding and catalytic steps. Recent work revealing the unusual behavior of the active site variant D58A of the representative PvuII endonuclease prompted speculation that the array of charged groups in the nuclease active site may also be linked to conformational behavior [Dupureur, C. M., and Conlan, L. H. (2000) Biochemistry 39, 10921-10927]. To address this issue, we analyzed the conformational behavior of active site variants of PvuII endonuclease using both NMR spectroscopic and thermodynamic methods. NMR spectroscopic analysis via (19)F and (1)H-(15)N HSQC experiments indicates that a number of side chain and backbone amide groups are perturbed upon Ala substitution at conserved active site residues Asp58, Glu68, and Lys70. Spectral changes are particularly pronounced for the lowest-activity mutants (D58A and K70A). These changes are accompanied by perturbations in conformational stability. Ala substitution at each of these positions results in 2-5 kcal/mol of stabilization over the wild-type enzyme at pH 7.7, changes which constitute increases in DeltaG(d)(H2O) of 20-50%. The pH dependencies of mutant enzyme stabilities are distinct from those of the wild type, results which confirm that these ionizable groups strongly influence stability. Wild-type enzyme stability is correlated with the ionization of groups shown to be important to metal ion binding and orientation. Correlations between spectral changes and conformational stability indicate that the latter measurements may prove useful in the evaluation of site-directed mutant restriction enzymes. More importantly, these results indicate that structure-function relationships in restriction enzyme active sites can be complex, and that the ensemble of conserved charged residues which mediate DNA hydrolysis in Mg(II)-dependent nucleases constitutes a critical link between function and conformation.     

Crystal structure of 3-chlorocatechol 1,2-dioxygenase key enzyme of a new modified ortho-pathway from the Gram-positive Rhodococcus opacus 1CP grown on 2-chlorophenol

The crystal structure of the 3-chlorocatechol 1,2-dioxygenase from the Gram-positive bacterium Rhodococcus opacus (erythropolis) 1CP, a Fe(III) ion-containing enzyme specialized in the aerobic biodegradation of 3-chloro- and methyl-substituted catechols, has been solved by molecular replacement techniques using the coordinates of 4-chlorocatechol 1,2-dioxygenase from the same organism (PDB code 1S9A) as a starting model and refined at 1.9 A resolution (R(free) 21.9%; R-factor 17.4%). The analysis of the structure and of the kinetic parameters for a series of different substrates, and the comparison with the corresponding data for the 4-chlorocatechol 1,2-dioxygenase isolated from the same bacterial strain, provides evidence of which active site residues are responsible for the observed differences in substrate specificity. Among the amino acid residues expected to interact with substrates, only three are altered Val53(Ala53), Tyr78(Phe78) and Ala221(Cys224) (3-chlorocatechol 1,2-dioxygenase(4-chlorocatechol 1,2-dioxygenase)), clearly identifying the substitutions influencing substrate selectivity in these enzymes. The crystallographic asymmetric unit contains eight subunits (corresponding to four dimers) that show heterogeneity in the conformation of a co-crystallized molecule bound to the catalytic non-heme iron(III) ion resembling a benzohydroxamate moiety, probably a result of the breakdown of recently discovered siderophores synthesized by Gram-positive bacteria. Several different modes of binding benzohydroxamate into the active site induce distinct conformations of the interacting protein ligands Tyr167 and Arg188, illustrating the plasticity of the active site origin of the more promiscuous substrate preferences of the present enzyme.     

The 'gating' residues Ile199 and Tyr326 in human monoamine oxidase B function in substrate and inhibitor recognition

The major structural difference between human monoamine oxidases A (MAO A) and B (MAO B) is that MAO A has a monopartite substrate cavity of ~550 ?(3) volume and MAO B contains a dipartite cavity structure with volumes of ~290 ?(3) (entrance cavity) and ~400 ?(3) (substrate cavity). Ile199 and Tyr326 side chains separate these two cavities in MAO B. To probe the function of these gating residues, Ile199Ala and Ile199Ala-Tyr326Ala mutant forms of MAO B were investigated. Structural data on the Ile199Ala MAO B mutant show no alterations in active site geometries compared with wild-type enzyme while the Ile199Ala-Tyr326Ala MAO B mutant exhibits alterations in residues 100-103 which are part of the loop gating the entrance to the active site. Both mutant enzymes exhibit catalytic properties with increased amine K(M) but unaltered k(cat) values. The altered K(M) values on mutation are attributed to the influence of the cavity structure in the binding and subsequent deprotonation of the amine substrate. Both mutant enzymes exhibit weaker binding affinities relative to wild-type enzyme for small reversible inhibitors. Ile199Ala MAO B exhibits an increase in binding affinity for reversible MAO B specific inhibitors which bridge both cavities. The Ile199Ala-Tyr326Ala double mutant exhibits inhibitor binding properties more similar to those of MAO A than to MAO B. These results demonstrate that the bipartite cavity structure in MAO B plays an important role in substrate and inhibitor recognition to distinguish its specificities from those of MAO A and provide insights into specific reversible inhibitor design for these membrane-bound enzymes.     

Crystal structure of enteropeptidase light chain complexed with an analog of the trypsinogen activation peptide.

Enteropeptidase is a membrane-bound serine protease that initiates the activation of pancreatic hydrolases by cleaving and activating trypsinogen. The enzyme is remarkably specific and cleaves after lysine residues of peptidyl substrates that resemble trypsinogen activation peptides such as Val-(Asp)4-Lys. To characterize the determinants of substrate specificity, we solved the crystal structure of the bovine enteropeptidase catalytic domain to 2.3 A resolution in complex with the inhibitor Val-(Asp)4-Lys-chloromethane. The catalytic mechanism and contacts with lysine at substrate position P1 are conserved with other trypsin-like serine proteases. However, the aspartyl residues at positions P2-P4 of the inhibitor interact with the enzyme surface mainly through salt bridges with the Nzeta atom of Lys99. Mutation of Lys99 to Ala, or acetylation with acetic anhydride, specifically prevented the cleavage of trypsinogen or Gly-(Asp)4-Lys-beta-naphthylamide and reduced the rate of inhibition by Val-(Asp)4-Lys-chloromethane 22 to 90-fold. For these reactions, Lys99 was calculated to account for 1.8 to 2.5 kcal mol(-1) of the free energy of transition state binding. Thus, a unique basic exosite on the enteropeptidase surface has evolved to facilitate the cleavage of its physiological substrate, trypsinogen.