Transition-state stabilization at the oxyanion binding sites of serine and thiol proteinases: hydrolyses of thiono and oxygen esters

X-ray diffraction studies suggested that the tetrahedral intermediate formed during the catalysis by serine and thiol proteinases can be stabilized by hydrogen bonds from the protein to the oxyanion of the intermediate [cf. Kraut, J. (1977) Annu. Rev. Biochem. 46, 331-358; Drenth, J., Kalk, K.H., & Swen, H.M. (1976) Biochemistry 15, 3731-3738]. To obtain evidence in favor or against this hypothesis, we synthesized thiono substrates (the derivatives of N-benzoyl-glycine methyl ester and N-acetylphenylalanine ethyl ester) containing a sulfur in place of the carbonyl oxygen atom of the scissile ester bond. We anticipated that this relatively subtle structural change specifically directed to the oxyanion binding site should produce serious catalytic consequences owing to the different properties of oxygen and sulfur if transition-state stabilization in the oxyanion hole is indeed important. In fact, while in alkaline hydrolysis the chemical reactivities of oxygen esters and corresponding thiono esters proved to be similar, neither chymotrypsin nor subtilisin hydrolyzed the thiono esters at a measurable rate. This result substantiates the crucial role of the oxyanion binding site in serine proteinase catalysis. On the basis of the similar values of the binding constants found for oxygen esters and their thiono counterparts, it can be concluded that the substitution of sulfur for oxygen significantly influences transition state stabilization but not substrate binding. The thiol proteinases papain and chymopapain react with the oxygen and thiono esters of N-benzoylglycine at similar rates. Apparently, in these reactions the above stabilizing mechanism is absent or not important, which is a major mechanistic difference between the catalyses by serine and thiol proteinases.     

The basic difference in catalyses by serine and cysteine proteinases resides in charge stabilization in the transition state

Besides the mechanistic similarities, in particular acylenzyme formation, kinetic investigations and X-ray diffraction studies have revealed some differences between the mechanisms of serine and cysteine proteinases: general base-catalysis in acylation, catalytic contribution by oxyanion binding, and a negatively charged catalytic triad in serine proteinases, but not in cysteine proteinases. In this paper we point out that all these differences are related and connected with the mode of stabilization of the zwitterionic species developing in the transition state of the reactions. In the case of serine proteinases this charge separation requires facilitation by the oxyanion binding and the negative charge of the catalytic triad. On the other hand cysteine proteinases do not require such contributions as they are capable of stabilizing the ion-pair even in the ground state of the reaction. Therefore, cysteine proteinases, in contrast to serine proteinases, may be regarded as "activated" enzymes.     

Molecular dynamics simulations of the acyl-enzyme and the tetrahedral intermediate in the deacylation step of serine proteases

Despite the availability of many experimental data and some modeling studies, questions remain as to the precise mechanism of the serine proteases. Here we report molecular dynamics simulations on the acyl-enzyme complex and the tetrahedral intermediate during the deacylation step in elastase catalyzed hydrolysis of a simple peptide. The models are based on recent crystallographic data for an acyl-enzyme intermediate at pH 5 and a time-resolved study on the deacylation step. Simulations were carried out on the acyl enzyme complex with His-57 in protonated (as for the pH 5 crystallographic work) and deprotonated forms. In both cases, a water molecule that could provide the nucleophilic hydroxide ion to attack the ester carbonyl was located between the imidazole ring of His-57 and the carbonyl carbon, close to the hydrolytic position assigned in the crystal structure. In the "neutral pH" simulations of the acyl-enzyme complex, the hydrolytic water oxygen was hydrogen bonded to the imidazole ring and the side chain of Arg-61. Alternative stable locations for water in the active site were also observed. Movement of the His-57 side-chain from that observed in the crystal structure allowed more solvent waters to enter the active site, suggesting that an alternative hydrolytic process directly involving two water molecules may be possible. At the acyl-enzyme stage, the ester carbonyl was found to flip easily in and out of the oxyanion hole. In contrast, simulations on the tetrahedral intermediate showed no significant movement of His-57 and the ester carbonyl was constantly located in the oxyanion hole. A comparison between the simulated tetrahedral intermediate and a time-resolved crystallographic structure assigned as predominantly reflecting the tetrahedral intermediate suggests that the experimental structure may not precisely represent an optimal arrangement for catalysis in solution. Movement of loop residues 216-223 and P3 residue, seen both in the tetrahedral simulation and the experimental analysis, could be related to product release. Furthermore, an analysis of the geometric data obtained from the simulations and the pH 5 crystal structure of the acyl-enzyme suggests that since His-57 is protonated, in some aspects, this crystal structure resembles the tetrahedral intermediate.     

The Oxyanion Hole in Serine beta-Lactamase Catalysis: Interactions of Thiono Substrates with the Active Site

Both functional and structural studies of serine beta-lactamases indicate the existence of an oxyanion hole at the active site with an important role in catalysis. The functional presence of the oxyanion hole is demonstrated by the previous observation that thiono-beta-lactams are very poor substrates of beta-lactamases (B. P. Murphy, and R. F. Pratt, 1988, Biochem. J. 256, 669-672) and in the present paper by the inability of these enzymes to catalyze hydrolysis of a thiono analog of a depsipeptide substrate. This thiono effect was first noted and interpreted in regard to classical serine hydrolases although the chemical basis for it has not been firmly established either in those enzymes or in beta-lactamases. In this paper a computational approach to a further understanding of the effect has been taken. The results for a class C beta-lactamase show that the deacylation tetrahedral intermediate interacted more strongly with the enzyme with an O(-) placed in the oxyanion hole than an S(-). On the other hand, the converse was true for acylation tetrahedral intermediate species, a result distinctly not in accord with experiment. These results indicate that the thiono effect does not arise from unfavorable interactions between enzyme and thiono substrate at the tetrahedral intermediate stage but must be purely kinetic in nature, i.e., arise in a transitional species at an early stage of the acylation reaction. The same conclusion as to the origin of the thiono effect was also indicated by a less extensive series of calculations on a class A beta-lactamase and on chymotrypsin.     

A serpin-induced extensive proteolytic susceptibility of urokinase-type plasminogen activator implicates distortion of the proteinase substrate-binding pocket and oxyanion hole in the serpin inhibitory mechanism.

The formation of stable complexes between serpins and their target serine proteinases indicates formation of an ester bond between the proteinase active-site serine and the serpin P1 residue [Egelund, R., Rodenburg, K.W., Andreasen, P.A., Rasmussen, M.S., Guldberg, R.E. & Petersen, T.E. (1998) Biochemistry 37, 6375-6379]. An important question concerning serpin inhibition is the contrast between the stability of the ester bond in the complex and the rapid hydrolysis of the acyl-enzyme intermediate in general serine proteinase-catalysed peptide bond hydrolysis. To answer this question, we used limited proteolysis to detect conformational differences between free urokinase-type plasminogen activator (uPA) and uPA in complex with plasminogen activator inhibitor-1 (PAI-1). Whereas the catalytic domain of free uPA, pro-uPA, uPA in complex with non-serpin inhibitors and anhydro-uPA in a non-covalent complex with PAI-1 was resistant to proteolysis, the catalytic domain of PAI-1-complexed uPA was susceptible to proteolysis. The cleavage sites for four different proteinases were localized in specific areas of the C-terminal beta-barrel of the catalytic domain of uPA, providing evidence that the serpin inhibitory mechanism involves a serpin-induced massive rearrangement of the proteinase active site, including the specificity pocket, the oxyanion hole, and main-chain binding area, rendering the proteinase unable to complete the normal hydrolysis of the acyl-enzyme intermediate. The distorted region includes the so-called activation domain, also known to change conformation on zymogen activation.     

The Oxyanion Hole in Serine β-Lactamase Catalysis: Interactions of Thiono Substrates with the Active Site

Both functional and structural studies of serine β-lactamases indicate the existence of an oxyanion hole at the active site with an important role in catalysis. The functional presence of the oxyanion hole is demonstrated by the previous observation that thiono-β-lactams are very poor substrates of β-lactamases (B. P. Murphy, and R. F. Pratt, 1988, Biochem. J.256, 669–672) and in the present paper by the inability of these enzymes to catalyze hydrolysis of a thiono analog of a depsipeptide substrate. This thiono effect was first noted and interpreted in regard to classical serine hydrolases although the chemical basis for it has not been firmly established either in those enzymes or in β-lactamases. In this paper a computational approach to a further understanding of the effect has been taken. The results for a class C β-lactamase show that the deacylation tetrahedral intermediate interacted more strongly with the enzyme with an O⁻ placed in the oxyanion hole than an S⁻. On the other hand, the converse was true for acylation tetrahedral intermediate species, a result distinctly not in accord with experiment. These results indicate that the thiono effect does not arise from unfavorable interactions between enzyme and thiono substrate at the tetrahedral intermediate stage but must be purely kinetic in nature, i.e., arise in a transitional species at an early stage of the acylation reaction. The same conclusion as to the origin of the thiono effect was also indicated by a less extensive series of calculations on a class A β-lactamase and on chymotrypsin.     

Benzoylamidoacetonitrile is bound as a thioimidate in the active site of papain

13C NMR spectroscopy has been used to demonstrate that 13CN-labeled benzoylamidoacetonitrile forms a covalent adduct with the thiol group of cysteine 25 in the active site of papain. Spectral comparison with model compounds indicates that the adduct is a thioimidate. On the basis of a proposed mechanism for the formation of the thioimidate, it is concluded that the -CH2C(= NH)S--imino nitrogen does not sit in the active site in the same manner as the thiol ester carbonyl oxygen of the thiol acyl enzyme (or the oxyanion of the tetrahedral intermediate). Thus, in this sense the stabilization of the thioimidate does not reflect a similarity in structure between the bound thioimidate and the transition state.     

Catalysis by dienelactone hydrolase: A variation on the protease mechanism

Dienelactone hydrolase (DLH), an enzyme from the β-ketoadipate pathway, catalyzes the hydrolysis of dienelactone to maleylacetate. Our inhibitor binding studies suggest that its substrate, dienelactone, is held in the active site by hydrophobic interactions around the lactone ring and by the ion pairs between its carboxylate and Arg-81 and Arg-206. Like the cysteine/serine proteases, DLH has a catalytic triad (Cys-123, His-202, Asp-171) and its mechanism probably involves the formation of covalently bound acyl intermediate via a tetrahedral intermediate. Unlike the proteases, DLH seems to protonate the incipient leaving group only after the collapse of the first tetrahedral intermediate, rendering DLH incapable of hydrolyzing amide analogues of its ester substrate. In addition, the triad His probably does not protonate the leaving group (enolate) or deprotonate the water for deacylation; rather, the enolate anion abstracts a proton from water and, in doing so, supplies the hydroxyl for deacylation. © 1993 Wiley-Liss, Inc.     

Mechanism of the reaction of papain with substrate-derived diazomethyl ketones. Implications for the difference in site specificity of halomethyl ketones for serine proteinases and cysteine proteinases and for stereoelectronic requirements in the papain catalytic mechanism.

The reactions of papain (EC 3.4.22.2) with substrate-derived diazomethyl ketones reported by Leary, Larsen, Watanabe & Shaw [Biochemistry (1977) 16, 5857--5861] are unusual in that (i) these reagents fail to react with low-molecular-weight thiols and (ii) the rate of reaction with the papain thiol group does not decrease to near-zero values across a pKa of 4 as the pH is decreased. Existing data are shown to suggest an interpretation involving neighbouring-group participation via transient thiohemiketal formation, rate-determining protonation by imidazolium ion and alkylation on sulphur via a three-membered cyclic transition state. Implications for (a) the difference in site-specificity exhibited by halomethyl ketones in their reactions with serine proteinases and cysteine proteinases and (b) stereoelectronic requirements in the mechanism of papain-catalysed hydrolysis are discussed. The possibility of two tetrahedral intermediates between adsorptive complex and acyl-enzyme is indicated.     

X-ray snapshots of serine protease catalysis reveal a tetrahedral intermediate

Studies on the catalytic mechanism and inhibition of serine proteases are widely used as paradigms for teaching enzyme catalysis. Ground-breaking work on the structures of chymotrypsin and subtilisin led to the idea of a conserved catalytic triad formed by the active site Ser, His and Asp residues. An oxyanion hole, consisting of the peptide amide of the active site serine and a neighbouring glycine, was identified, and hydrogen bonding in the oxyanion hole was suggested to stabilize the two proposed tetrahedral intermediates on the catalytic pathway. Here we show electron density changes consistent with the formation of a tetrahedral intermediate during the hydrolysis of an acyl-enzyme complex formed between a natural heptapeptide and elastase. No electron density for an enzyme-product complex was observed. The structures also suggest a mechanism for the synchronization of hydrolysis and peptide release triggered by the conversion of the sp2 hybridized carbonyl carbon to an sp3 carbon in the tetrahedral intermediate. This affects the location of the peptide in the active site cleft, triggering the collapse of a hydrogen bonding network between the peptide and the beta-sheet of the active site.     

A mechanistic view of enzyme inhibition and peptide hydrolysis in the active site of the SARS-CoV 3C-like peptidase

The 3C-like main peptidase 3CL(pro) is a viral polyprotein processing enzyme essential for the viability of the Severe Acute Respiratory Syndrome coronavirus (SARS-CoV). While it is generalized that 3CL(pro) and the structurally related 3C(pro) viral peptidases cleave their substrates via a mechanism similar to that underlying the peptide hydrolysis by chymotrypsin-like serine proteinases (CLSPs), some of the hypothesized key intermediates have not been structurally characterized. Here, we present three crystal structures of SARS 3CL(pro) in complex with each of two members of a new class of peptide-based phthalhydrazide inhibitors. Both inhibitors form an unusual thiiranium ring with the nucleophilic sulfur atom of Cys145, trapping the enzyme's catalytic residues in configurations similar to the intermediate states proposed to exist during the hydrolysis of native substrates. Most significantly, our crystallographic data are consistent with a scenario in which a water molecule, possibly via indirect coordination from the carbonyl oxygen of Thr26, has initiated nucleophilic attack on the enzyme-bound inhibitor. Our data suggest that this structure resembles that of the proposed tetrahedral intermediate during the deacylation step of normal peptidyl cleavage.     

Role of catalytic residues in the formation of a tetrahedral adduct in the acylation reaction of bovine β-trypsin: A molecular orbital study

In the acylation reaction of serine proteases the effect of amino acid residues on the geometrical change of the catalytic site from Michaelis to tetrahedral state was studied by using ab initio molecular orbital calculations. Amino acid residues in the catalytic site and the peptide substrate were calculated as a quantum mechanical region, and all the other amino acid residues and the calcium ion were included in the calculation as the electrostatic effects. The effects of Asp102, Asp194, N-terminus and the oxyanion binding site are large. The oxyanion binding site directly stabilizes the tetrahedral substrate. Asp102 stabilizes the enzyme intermediate, interacting with the protonated His57 residue. In order to elucidate the roles of Asp102 and the oxyanion binding site, energy decomposition analyses were done for the intermolecular interactions. The contribution of Asp102 and the oxyanion binding site to the decrease of energy in the geometrical change is due to the electrostatic effect. The energies of the proton shuttle from Ser195 O^γ to the leaving group of the substrate were calculated for amide and ester substrate models.     

Differences in midgut serine proteinases from larvae of the bruchid beetles Callosobruchus maculatus and Zabrotes subfasciatus

Proteinase activities in the larval midguts of the bruchids Callosobruchus maculatus and Zabrotes subfasciatus were investigated. Both midgut homogenates showed a slightly acidic to neutral pH optima for the hydrolysis of fluorogenic substrates. Proteolysis of epsilon-aminocaproil-Leu-Cys(SBzl)-MCA was totally inhibited by the cysteine proteinase inhibitors E-64 and leupeptin, and was activated by 1.5 mM DTT in both insects, while hydrolysis of the substrate Z-ArgArg-MCA was inhibited by aprotinin and E-64, which suggests that it is being hydrolysed by serine and cysteine proteinases. Gel assays showed that the proteolytic activity in larval midgut of C. maculatus was due to five major cysteine proteinases. However, based on the pattern of E-64 and aprotinin inhibition, proteolytic activity in larval midgut of Z. subfasciatus was not due only to cysteine proteinases. Fractionation of the larval midgut homogenates of both bruchids through ion-exchange chromatography (DEAE-Sepharose) revealed two peaks of activity against Z-ArgArg-MCA for both bruchid species. The fractions from C. maculatus have characteristics of cysteine proteinases, while Z. subfasciatus has one non-retained peak of activity containing cysteine proteinases and another eluted in a gradient of 250-350 mM NaCl. The proteolytic activity of the retained peak is higher at pH 8.8 than at pH 6.0 and corresponds with a single peak that is active against N-p-tosyl-GlyGlyArg-MCA, and sensitive to 250 microM aprotinin (90% inhibition). The peak contains a serine proteinase which hydrolyzes alpha-amylase inhibitor 1 from the common bean (Phaseolus vulgaris). Arch.     

Probing the Ser-Ser-Lys catalytic triad mechanism of peptide amidase: computational studies of the ground state, transition state, and intermediate

Peptide amidase (Pam), a hydrolytic enzyme that belongs to the amidase signature (AS) family, selectively catalyzes the hydrolysis of the C-terminal amide bond (CO-NH(2)) of peptides. The recent availability of the X-ray structures of Pam, fatty acid amide hydrolase, and malonamidase E2 has led to the proposal of a novel Ser-Ser-Lys catalytic triad mechanism for the amide hydrolysis by the AS enzymes. The molecular dynamics (MD) simulations using the CHARMM force field were performed to explore the catalytic mechanism of Pam. The 1.8 A X-ray crystal structure of Pam in complex with the amide analogue of chymostatin was chosen for the initial coordinates for the MD simulations. The five systems that were investigated are as follows: (i) enzyme.substrate with Lys123-NH(2), (ii) enzyme.substrate with Lys123-NH(3)(+), (iii) enzyme.substrate with Lys123-NH(3)(+) and Ser226-O(-), (iv) enzyme.transition state, and (v) enzyme.tetrahedral intermediate. Our data support the presence of the hydrogen bonding network among the catalytic triad residues, Ser226, Ser202, and Lys123, where Ser226 acts as the nucleophile and Ser202 bridges Ser226 and Lys123. The MD simulation supports the catalytic role of the crystallographic waters, Wat1 and Wat2. In all the systems that have been studied, the backbone amide nitrogens of Asp224 and Thr223 create an oxyanion hole by hydrogen bonding to the terminal amide oxygen of the substrate, and stabilize the oxyanion tetrahedral intermediate. The results from both our computational investigation and previously published experimental pH profile support two mechanisms. In a mechanism that is relevant at lower pH, the Lys123-NH(3)(+)-Ser202 dyad provides structural support to the catalytic residue Ser226, which in turn carries out a nucleophilic attack at the substrate amide carbonyl in concert with Wat1-mediated deprotonation and stabilization of the tetrahedral transition state by the oxyanion hole. In the mechanism operating at higher pH, the Lys123-NH(2)-Ser202 catalytic dyad acts as a general base to assist addition of Ser226 to the substrate amide carbonyl. The results from the MD simulation of the tetrahedral intermediate state show that both Ser202 and Lys123 are possible candidates for protonation of the leaving group, NH(2), to form the acyl-enzyme intermediate.     

Direct observation by X-ray analysis of the tetrahedral "intermediate" of aspartic proteinases.

We report the X-ray analysis at 2.0 A resolution for crystals of the aspartic proteinase endothiapepsin (EC 3.4.23.6) complexed with a potent difluorostatone-containing tripeptide renin inhibitor (CP-81,282). The scissile bond surrogate, an electrophilic ketone, is hydrated in the complex. The pro-(R) (statine-like) hydroxyl of the tetrahedral carbonyl hydrate is hydrogen-bonded to both active-site aspartates 32 and 215 in the position occupied by a water in the native enzyme. The second hydroxyl oxygen of the hydrate is hydrogen-bonded only to the outer oxygen of Asp 32. These experimental data provide a basis for a model of the tetrahedral intermediate in aspartic proteinase-mediated cleavage of the amide bond. This indicates a mechanism in which Asp 32 is the proton donor and Asp 215 carboxylate polarizes a bound water for nucleophilic attack. The mechanism involves a carboxylate (Asp 32) that is stabilized by extensive hydrogen bonding, rather than an oxyanion derivative of the peptide as in serine proteinase catalysis.     

Direct crystallographic observation of an acyl-enzyme intermediate in the elastase-catalyzed hydrolysis of a peptidyl ester substrate: Exploiting the "glass transition" in protein dynamics

The crystal structure of the acyl complex of porcine pancreatic elastase with its peptidyl ester substrate N-acetyl-ala-ala-ala-methyl ester (Ac(Ala)3OMe) has been determined at 2.5 A resolution. The complex was stabilized by exploiting the "glass transition" in protein dynamics that occurs at around -53 degrees C (220 K). Substrate was flowed into the crystal in a cryoprotective solvent above this temperature, and then the crystal was rapidly cooled to a temperature below the transition to trap the species that formed. The use of a flow cell makes the experiment a kinetic one and means that the species prior to the rate determining transition state has a chance to accumulate. The resulting crystal structure shows an acyl-enzyme intermediate in which the leaving group is absent and the carbonyl carbon of the C-terminal alanine residue is covalently bound to the gamma oxygen of the active site serine. The ester carbonyl shows no significant distortion from planarity, with the carbonyl oxygen forming one hydrogen bond with the oxyanion hole. The tripeptide is bound in an extended antiparallel beta-sheet with main chain residues of the enzyme. The geometry and interactions of this acyl-enzyme suggest that it represents a productive intermediate. To test this hypothesis, the same crystal was then warmed above the glass transition temperature and a second data set was collected. The resulting electron density map shows no sign of the substrate, indicating hydrolysis of the intermediate followed by product release. This experiment provides direct evidence for the importance of dynamic properties in catalysis and also provides a blueprint for the stabilization of other short-lived species for direct crystallographic observation.     

The importance of a distal hydrogen bonding group in stabilizing the transition state in subtilisin BPN'

Stabilization of an oxyanion transition state is important to catalysis of peptide bond hydrolysis in all proteases. For subtilisin BPN', a bacterial serine protease, structural data suggest that two hydrogen bonds stabilize the tetrahedral-like oxyanion intermediate: one from the main chain NH of Ser221 and another from the side chain NH2 of Asn155. Molecular dynamic studies (Rao, S., N., Singh, U., C. Bush, P. A., and Kollman, P. A. (1987) Nature 328, 551-554) have indicated the gamma-hydroxyl of Thr220 may be a third hydrogen bond donor even though it is 4A away in the static x-ray structure. We have probed the role of Thr220 by replacing it with serine, cysteine, valine, or alanine by site-directed mutagenesis. These substitutions were intended to alter the size and hydrogen bonding ability of residue 220. Removal of the gamma-hydroxyl group reduced the transition state stabilization energy (delta delta GT) by 1.8-2.1 kcal/mol depending upon the substitution. By comparison, removal of the gamma-methyl group in the Thr220 to serine mutation only decreased delta GT by 0.5 kcal/mol. The gamma-hydroxyl of Thr220 is most important for catalysis, not substrate binding, because virtually all of the effects were on kcat and not KM. The role of the Thr220 hydroxyl is functionally independent from the amide NH2 of Asn155 because the free energy effects of double alanine mutants at these two positions are additive. These data indicate that a distal hydrogen bond donor, namely the hydroxyl of Thr220, plays a functionally important role in stabilizing the oxyanion transition state in subtilisin which is independent of Asn155.     

Crystal structures of alpha-lytic protease complexes with irreversibly bound phosphonate esters

The structures of the complexes with alpha-lytic protease of both phosphorus stereoisomers of N-[(2S)-2-[[[(1R)-1-[N-[(tert-butyloxycarbonyl)-L-alanyl-L-alanyl- L-prolyl]amino]-2-methylpropyl]-phenoxyphosphinyl]oxy]propanoyl]- L-alanine methyl ester, an analogue of the peptide Boc-Ala-Ala-Pro-Val-Ala-Ala where Val is replaced with an analogous phosphonate phenyl ester and the subsequent Ala is replaced with lactate, have been determined to high resolution (1.9 A) by X-ray crystallography. Both stereoisomers inactivate the enzyme but differ by a factor of 2 in the second-order rate constant for inactivation [Sampson, N. S., & Bartlett, P. A. (1991) Biochemistry (preceding paper in this issue)]. One isomer (B) forms a tetrahedral adduct in which the phosphonate phenyl ester is displaced by the active site serine (S195) and interacts with the enzyme across seven substrate recognition sites that span both sides of the scissile bond. Seven hydrogen bonds are formed with the enzyme, and 510 A2 of hydrophobic surface area is buried when the inhibitor interacts with the enzyme. Although two hydrogen bonds are gained by incorporation of two residues on the C-terminal side of the scissile bond into the inhibitor, there is very little adjustment in the structure of the enzyme in this region. Surprisingly, the active site histidine (H57) does not interact with the phosphonate, apparently because the phosphonate lacks negative charge in or near the oxyanion hole, and instead, the side chain rotates out of the active site cleft and hydrogen bonds with solvent. The other isomer (A) forms a mixture of two different tetrahedral adducts in the active site, both covalently bonded to Ser 195. One adduct, at approximately 58% occupancy, is exactly the same in structure as the complex formed with isomer B, and the other adduct, at 42% occupancy, has lost the two residues C-terminal to the scissile bond by hydrolysis. In the lower occupancy structure, His 57 does not rotate out of the active site and forms a hydrogen bond with the phosphonate oxygen instead. The structures of both complexes were insensitive to pH. As very little change in structure accompanies the histidine rotation, the complex with isomer B provides an excellent mimic for the structure of the transition state (or high-energy reaction intermediate) that spans both sides of the scissile bond.     

Proteinase activities in total extracts and in medium conditioned by Acanthamoeba polyphaga trophozoites

Acanthamoeba species can cause granulomatous encephalitis and keratitis in man. The mechanisms that underlie tissue damage and invasion by the amoebae are poorly understood, but involvement of as yet uncharacterized proteinases has been suggested. Here, we employed gelatin-containing gels and azocasein assays to examine proteinase activities in cell lysates and in medium conditioned by Acanthamoeba polyphaga trophozoites. Azocasein hydrolysis by cell lysates was optimally detected at pH 4.0-5.0 and was predominantly associated with the activity of cysteine proteinases. Compatible with enzyme activation during secretion, culture supernatants additionally contained a prominent azocasein hydrolyzing activity attributable to serine proteinases; these enzymes were better detected at pH 6.0 and above, and resolved at 47, 60, 75, 100, and >110 kDa in overlay gelatin gels. Although a similar banding profile was observed in gels of trophozoite lysates, intracellular serine proteinases were shown to be activated during electrophoresis and to split the substrate during migration in sodium dodecyl sulfate gels. Blockage of serine proteinases with phenylmethylsulfonylfluoride prior to electrophoresis permitted the detection of 43-, 59-, 70-, and 100-130-kDa acidic cysteine proteinases in cell lysates, and of 3 (43, 70, and 130 kDa) apparently equivalent enzymes in culture supernatants. Under the conditions employed, no band associated with a metalloproteinase activity could be depicted in substrate gels, although the discrete inhibition of supernatants' azocaseinolytic activity by 1,10-phenanthroline suggested secretion of some metalloproteinase.     

Physiological adaptation explains the insensitivity of Baris coerulescens to transgenic oilseed rape expressing oryzacystatin I

Larvae of Baris coerulescens Scop. (Coleoptera: Curculionidæ) exhibit a complex array of gut proteinase activities comprising cysteine and serine proteinases. The major cysteine proteinase activity, showing an optimum at pH 6.0, corresponds to at least 4 different proteinases. On the contrary, the minor serine proteinase activity, with an optimum at pH 9.0, seems to be due essentially to a single proteinase. The cysteine proteinase inhibitor oryzacystatin I (OC-I) inhibits completely the cysteine proteinase activity in vitro. However, larval growth and survival were not significantly different on control and transgenic oilseed rape plants expressing high levels of active OC-I. In larvae grown on transgenic plants, cysteine proteinase activity was dramatically decreased, whereas serine proteinase activity was increased by more than 2-fold, when compared to larvae raised on control plants. For both activities, no new proteinase was detected in insects fed plants expressing OC-I. These results suggest that partial compensation of the inhibition of cysteine proteinase activity by the increase in serine proteinase activity allowed the larvae to overcome the effects of OC-I consumption. This case illustrates problems that could arise when trying to achieve high levels of protection for plants against Coleopteran pests possessing a complex digestive proteinase pool.