Plasmepsin II, an acidic hemoglobinase from the Plasmodium falciparum food vacuole, is active at neutral pH on the host erythrocyte membrane skeleton.

Plasmepsin II, an aspartic protease from the human intraerythrocytic parasite Plasmodium falciparum, is involved in degradation of the host cell hemoglobin within the acidic food vacuole of the parasite. Previous characterization of enzymatic activities from Plasmodium soluble extracts, responsible for in vitro hydrolysis of erythrocyte spectrin, had shown that the hydrolysis process occurred at pH 5.0 and involved aspartic protease(s) cleaving mainly within the SH3 motif of the spectrin alpha-subunit. Therefore, we used a recombinant construct of the erythroid SH3 motif as substrate to investigate the involvement of plasmepsins in spectrin hydrolysis. Using specific anti-plasmepsin II antibodies in Western blotting experiments, plasmepsin II was detected in chromatographic fractions enriched in the parasite SH3 hydrolase activity. Involvement of plasmepsin II in hydrolysis was demonstrated by mass spectrometry identification of cleavage sites in the SH3 motif, upon hydrolysis by Plasmodium extract enzymatic activity, and by recombinant plasmepsin II. Furthermore, recombinant plasmepsin II digested native spectrin at pH 6.8, either purified or situated in erythrocyte ghosts. Additional degradation of actin and protein 4.1 from ghosts was observed. Specific antibodies were used in confocal imaging of schizont-infected erythrocytes to localize plasmepsin II in mature stages of the parasite development cycle; antibodies clearly labeled the periphery of the parasites. Taken together, these results strongly suggest that, in addition to hemoglobin degradation, plasmepsin II might be involved in cytoskeleton cleavage of infected erythrocytes.     

New inhibitors of the malaria aspartyl proteases plasmepsin I and II

New inhibitors of plasmepsin I and II, the aspartic proteases of the malaria parasite Plasmodium falciparum, are described. From paralell solution phase chemistry, several reversed-statine type isostere inhibitors, many of which are aza-peptides, have been prepared. The synthetic strategy delivers the target compounds in good to high overall yields and with excellent stereochemical control throughout the developed route. The final products were tested for their plasmepsin I and II inhibiting properties and were found to exhibit modest but promising activity. The best inhibitor exhibits K(i) values of 250 nM and 1.4 microM for Plm I and II, respectively.     

Solid-phase library synthesis of reversed-statine type inhibitors of the malarial aspartyl proteases plasmepsin I and II

With the aim to develop inhibitors of the plasmepsin I and II aspartic proteases of the malaria parasite Plasmodium falciparum, we have synthesized sets of libraries from novel reversed-statine isosteres, using a combination of solution phase and solid phase chemistry. The synthetic strategy furnishes the library compounds in good to high overall yields and with excellent stereochemical control throughout the developed route. The products were evaluated for their plasmepsin I and II inhibiting properties and were found to exhibit modest but promising activity. The best inhibitor exhibits an in vitro activity of 28% inhibition of plasmepsin II at an inhibitor concentration of 0.5 microM (K(i) for Plm II=5.4 microM).     

α-Substituted norstatines as the transition-state mimic in inhibitors of multiple digestive vacuole malaria aspartic proteases

The impact of moving the P1 side-chain from the β-position to the α-position in norstatine-containing plasmepsin inhibitors was investigated, generating two new classes of tertiary alcohol-comprising α-benzylnorstatines and α-phenylnorstatines. Twelve α-substituted norstatines were designed, synthesized and evaluated for their inhibitory potencies against plasmepsin II and the plasmepsin IV orthologues (PM4) present in the digestive vacuole of all four Plasmodium species causing malaria in man. New synthetic routes were developed for producing the desired α-substituted norstatines as pure stereoisomers. The best compounds provided K_i values in the nanomolar range for all PM4, with a best value of 110 nM in PM4 from Plasmodium ovale. In addition, excellent selectivity over the closely related human aspartic protease Cathepsin D was achieved. The loss of affinity to Plasmodium falciparum PM4, which was experienced upon the move of the P1 substituent, was rationalized by the calculation of inhibitor–protein binding affinities using the linear interaction energy method (LIE).     

Mapping selectivity and specificity of active site of plasmepsins from Plasmodium falciparum using molecular interaction field approach

In the search for selectivity, the aspartic proteases are known to be a very difficult case because the enzymes of this family are not only sequentially but structurally also very similar. To gain insight into the selectivity and specificity of the aspartic proteases family we characterized the binding sites of four malarial aspartic protease (plasmepsin I, plasmepsin II, plasmepsin IV, P. vivax plasmepsin) and two human aspartic proteases (cathepsin D and pepsin) with the intention of identifying the regions that could be potential sites for obtaining selectivity using molecular interaction field approach.     

Identification and characterization of allophenylnorstatine-based inhibitors of plasmepsin II, an antimalarial target

Plasmepsin II is a key enzyme in the life cycle of the Plasmodium parasites responsible for malaria, a disease that afflicts more than 300 million individuals annually. Since plasmepsin II inhibition leads to starvation of the parasite, it has been acknowledged as an important target for the development of new antimalarials. In this paper, we identify and characterize high-affinity inhibitors of plasmepsin II based upon the allophenylnorstatine scaffold. The best compound, KNI-727, inhibits plasmepsin II with a K(i) of 70 nM and a 22-fold selectivity with respect to the highly homologous human enzyme cathepsin D. KNI-727 binds to plasmepsin II in a process favored both enthalpically and entropically. At 25 degrees C, the binding enthalpy (DeltaH) is -4.4 kcal/mol and the entropic contribution (-TDeltaS) to the Gibbs energy is -5.56 kcal/mol. Structural stability measurements of plasmepsin II were also utilized to characterize inhibitor binding. High-sensitivity differential scanning calorimetry experiments performed in the absence of inhibitors indicate that, at pH 4.0, plasmepsin II undergoes thermal denaturation at 63.3 degrees C. The structural stability of the enzyme increases with inhibitor concentration in a manner for which the binding energetics of the inhibitor can quantitatively account. The effectiveness of the best compounds in killing the malaria parasite was validated by performing cytotoxicity assays in red blood cells infected with Plasmodium falciparum. EC50s ranging between 6 and 10 microM (3-6 microg/mL) were obtained. These experiments demonstrate the viability of the allophenylnorstatine scaffold in the design of powerful and selective plasmepsin inhibitors.     

New potent C2-symmetric malaria plasmepsin I and II inhibitors

A series of malaria plasmepsin (Plm) I and II inhibitors containing a C(2)-symmetric core structure have been synthesised and tested for protease inhibition activity. These compounds can be prepared using a straightforward synthesis involving a phenol nucleophilic ring opening of a diepoxide. Exemplar compounds synthesised exhibited remarkable inhibitory activity against both Plm I and II, notably 15c with K(i) values of 2.7nM and 0.25nM respectively, as well as showing >100-fold selectivity against Cathepsin D.     

Computational analysis of plasmepsin IV bound to an allophenylnorstatine inhibitor

The plasmepsin proteases from the malaria parasite Plasmodium falciparum are attracting attention as putative drug targets. A recently published crystal structure of Plasmodium malariae plasmepsin IV bound to an allophenylnorstatine inhibitor [Clemente, J.C. et al. (2006) Acta Crystallogr. D 62, 246-252] provides the first structural insights regarding interactions of this family of inhibitors with plasmepsins. The compounds in this class are potent inhibitors of HIV-1 protease, but also show nM binding affinities towards plasmepsin IV. Here, we utilize automated docking, molecular dynamics and binding free energy calculations with the linear interaction energy LIE method to investigate the binding of allophenylnorstatine inhibitors to plasmepsin IV from two different species. The calculations yield excellent agreement with experimental binding data and provide new information regarding protonation states of active site residues as well as conformational properties of the inhibitor complexes.     

Processing, activity, and inhibition of recombinant cyprosin, an aspartic proteinase from cardoon (Cynara cardunculus).

The cDNA encoding the precursor of an aspartic proteinase from the flowers of the cardoon, Cynara cardunculus, was expressed in Pichia pastoris, and the recombinant, mature cyprosin that accumulated in the culture medium was purified and characterized. The resultant mixture of microheterogeneous forms was shown to consist of glycosylated heavy chains (34 or 32 kDa) plus associated light chains with molecular weights in the region of 14,000-18,000, resulting from excision of most, but not all, of the 104 residues contributed by the unique region known as the plant specific insert. SDS-polyacrylamide gel electrophoresis under non-reducing conditions indicated that disulfide bonding held the heavy and light chains together in the heterodimeric enzyme forms. In contrast, when a construct was expressed in which the nucleotides encoding the 104 residues of the plant specific insert were deleted, the inactive, unprocessed precursor form (procyprosin) accumulated, indicating that the plant-specific insert has a role in ensuring that the nascent polypeptide is folded properly and rendered capable of being activated to generate mature, active proteinase. Kinetic parameters were derived for the hydrolysis of a synthetic peptide substrate by wild-type, recombinant cyprosin at a variety of pH and temperature values and the subsite requirements of the enzyme were mapped using a systematic series of synthetic inhibitors. The significance is discussed of the susceptibility of cyprosin to inhibitors of human immunodeficiency virus proteinase and particularly of renin, some of which were found to have subnanomolar potencies against the plant enzyme.     

New benzimidazole derivatives as antiplasmodial agents and plasmepsin inhibitors: synthesis and analysis of structure-activity relationships

The newly synthesized benzimidazole compounds were suggested to be inhibitors of Plasmodium falciparum plasmepsin II and human cathepsin D by virtual screening of an internal library of synthetic compounds. This was confirmed by enzyme inhibition studies that gave IC(50) values in the low micromolar range (2-48μM). Ligand docking studies with plasmepsin II predicted binding of benzimidazole compounds at the center of the extended substrate-binding cleft. According to the plausible mode of binding, the pyridine ring of benzimidazole compounds interacted with S1' subsite residues whereas the acetophenone moiety was in contact with S1-S3 subsites of plasmepsin II active center. The benzimidazole derivatives were evaluated for capacity to inhibit the growth of intraerythrocytic P. falciparum in culture. Four benzimidazole compounds inhibited parasite growth at ?3μM. The most active compound 10, 1-(4-phenylphenyl)-2[2-(pyridinyl-2-yl)-1,3-benzdiazol-1-yl]ethanone showed an IC(50) of 160nM. The substitution of a phenyl group and a chlorine atom at the para position of the acetophenone moiety were shown to be crucial for antiplasmodial activity.     

Characterization of full length and truncated type I human methionine aminopeptidases expressed from Escherichia coli

Methionine aminopeptidase (MetAP) carries out an essential posttranslational modification of nascent proteins by removing the initiator methionine and is recognized as a potential target for developing antibacterial, antifungal, and anticancer agents. We have established an Escherichia coli expression system for human type I MetAP (HsMetAP1) and characterized the full length HsMetAP1 and its N-terminal-truncated mutants HsMetAP1(Delta1-66) and HsMetAP1(Delta1-135) for hydrolysis of several thiopeptolide and peptide substrates and inhibition by a series of nonpeptidic inhibitors. Although the N-terminal extension with zinc finger motifs in HsMetAP1 is not required for enzyme activity, it has a significant impact on the interaction of the enzyme with substrates and inhibitors. In hydrolysis of the thiopeptolide substrates, a relaxation of stringent specificity for the terminal methionine was observed in the truncated mutants. However, this relaxation of specificity was not detectable in hydrolysis of tripeptide or tetrapeptide substrates. Several nonpeptidic inhibitors showed potent inhibition of the mutant HsMetAP1(Delta1-66) but exhibited only weak or no inhibition of the full length enzyme. With the recombinant HsMetAP1 available, we have identified several MetAP inhibitors with submicromolar inhibitory potencies against E. coli MetAP (EcMetAP1) that do not affect HsMetAP1. These results have demonstrated the possibility of developing MetAP inhibitors as antibacterial agents with minimum human toxicity. In addition, micromolar inhibitors of HsMetAP1 identified in this study can serve as tools for investigating the functions of HsMetAP1 in physiological and pathological processes.     

Increased glycosidic bond stabilities in 4-C-hydroxymethyl linked disaccharides

Three new hydroxymethyl-linked non-natural disaccharide analogues, containing an additional methylene group in between the glycosidic linkage, were synthesized by utilizing 4-C-hydroxymethyl-α-d-glucopyranoside as the glycosyl donor. A kinetic study was undertaken to assess the hydrolytic stabilities of these new disaccharide analogues toward acid-catalyzed hydrolysis, at 60 °C and 70 °C. The studies showed that the disaccharide analogues were stable, by an order of magnitude, than naturally-occurring disaccharides, such as, cellobiose, lactose, and maltose. The first order rate constants were lower than that of methyl glycosides and the trend of hydrolysis rate constants followed that of naturally-occurring disaccharides. α-Anomer showed faster hydrolysis than the β-anomer and the presence of axial hydroxyl group also led to faster hydrolysis among the disaccharide analogues. Energy minimized structures, derived through molecular modeling, showed that dihedral angles around the glycosidic bond in disaccharide analogues were nearly similar to that of naturally-occurring disaccharides.     

Kinetics of the inhibition of neutrophil proteinases by recombinant elafin and pre-elafin (trappin-2) expressed in Pichia pastoris.

Elafin and its precursor, trappin-2 or pre-elafin, are specific endogenous inhibitors of human neutrophil elastase and proteinase 3 but not of cathepsin G. Both inhibitors belong, together with secretory leukocyte protease inhibitor, to the chelonianin family of canonical protease inhibitors of serine proteases. A cDNA coding either elafin or its precursor, trappin-2, was fused in frame with yeast alpha-factor cDNA and expressed in the Pichia pastoris yeast expression system. Full-length elafin or full-length trappin-2 were secreted into the culture medium with high yield, indicating correct processing of the fusion proteins by the yeast KEX2 signal peptidase. Both recombinant inhibitors were purified to homogeneity from concentrated culture medium by one-step cationic exchange chromatography and characterized by N-terminal amino acid sequencing, Western blot and kinetic studies. Both recombinant elafin and trappin-2 were found to be fast-acting inhibitors of pancreatic elastase, neutrophil elastase and proteinase 3 with k(ass) values of 2-4 x 10(6) m(-1).s(-1), while dissociation rate constants k(diss) were found to be in the 10(-4) s(-1) range, indicating low reversibility of the complexes. The equilibrium dissociation constant K(i) for the interaction of both recombinant inhibitors with their target enzymes was either directly measured for pancreatic elastase or calculated from k(ass) and k(diss) values for neutrophil elastase and proteinase 3. K(i) values were found to be in the 10(-10) molar range and virtually identical for both inhibitors. Based on the kinetic parameters determined here, it may be concluded that both recombinant elafin and trappin-2 may act as potent anti-inflammatory molecules and may be of therapeutic potential in the treatment of various inflammatory lung diseases.     

The integration of genomic and structural information in the development of high affinity plasmepsin inhibitors

The plasmepsins are key enzymes in the life cycle of the Plasmodium parasites responsible for malaria. Since plasmepsin inhibition leads to parasite death, these enzymes have been acknowledged to be important targets for the development of new antimalarial drugs. The development of effective plasmepsin inhibitors, however, is compounded by their genomic diversity which gives rise not to a unique target for drug development but to a family of closely related targets. Successful drugs will have to inhibit not one but several related enzymes with high affinity. Structure-based drug design against heterogeneous targets requires a departure from the classic 'lock-and-key' paradigm that leads to the development of conformationally constrained molecules aimed at a single target. Drug molecules designed along those principles are usually rigid and unable to adapt to target variations arising from naturally occurring genetic polymorphisms or drug-induced resistant mutations. Heterogeneous targets need adaptive drug molecules, characterised by the presence of flexible elements at specific locations that sustain a viable binding affinity against existing or expected polymorphisms. Adaptive ligands have characteristic thermodynamic signatures that distinguish them from their rigid counterparts. This realisation has led to the development of rigorous thermodynamic design guidelines that take advantage of correlations between the structure of lead compounds and the enthalpic and entropic components of the binding affinity. In this paper, we discuss the application of the thermodynamic approach to the development of high affinity (K(i) - pM) plasmepsin inhibitors. In particular, a family of allophenylnorstatine-based compounds is evaluated for their potential to inhibit a wide spectrum of plasmepsins.     

Human blood platelet 3': 5'-cyclic nucleotide phosphodiesterase. Isolation of low-Km and high-Km phosphodiesterase.

Human blood platelet contained at least three kinetically distinct forms of 3': 5'-cyclic nucleotide phosphodiesterase (3': 5'-cyclic-AMP 5'-nucleotidohydrolase, EC 3.1.4.17) (F I, F II, and F III) which were clearly separated by DEAE-cellulose column chromatography. Although a few properties of the platelet phosphodiesterases such as their substrate affinities and DEAE-cellulose profile resembled somewhat those of the three 3': 5'-cyclic nucleotide phosphodiesterase in rat liver reported by Russell et al. [10], there were pronounced differences in some properties between the platelet and the liver enzymes: (1) the platelet enzymes hydrolyzed both cyclic nucleotides and lacked a highly specific cyclic guanosine 3': 5'-monophosphate (cyclic GMP) phosphodiesterase and (2) kinetic data of the platelet enzymes indicated that cyclic adenosine 3': 5'-monophosphate (cyclic AMP) and cyclic GMP interact with a single catalytic site on the enzyme. F I was a cyclic nucleotide phosphodiesterase with a high Km for cyclic AMP and a negatively cooperative low Km for cyclic GMP. F II hydrolyzed cyclic AMP and cyclic GMP about equally with a high Km for both substrates. F III was low Km phosphodiesterase which hydrolyzed cyclic AMP faster than cyclic GMP. Each cyclic nucleotide acted as a competitive inhibitor of the hydrolysis of the other nucleotide by these three fractions with Ki values similar to the Km values for each nucleotide suggesting that the hydrolysis of both cyclic AMP and cyclic GMP was catalyzed by a single catalytic site on the enzyme. However, cyclic GMP at low concentration (below 10 muM) was an activator of cyclic AMP hydrolysis by F I. Papaverine and EG 626 acted as competitive inhibitors of each fraction with virtually the same Ki value in both assays using either cyclic AMP or cyclic GMP as the substrate. The ratio of cyclic AMP hydrolysis to cyclic GMP hydrolysis by each fraction did not vary significantly after freezing/thawing or heat treatment. These facts also suggest that both nucleotides were hydrolyzed by the same catalytic site on the enzyme. The differences in apparent Ki values for inhibitors such as cyclic nucleotides, papaverine and EG 626 would indicate that three enzymes were different from each other. Centrifugation in a continuous sucrose gradient revealed sedimentation coefficients F I and II had 8.9 S and F III 4.6 S. The molecular weight of these forms, determined by gel filtration on a Sepharose 6B column, were approx. 240 000 (F I and II) and 180 000 (F III). F III was purified extensively (70-fold) from homogenate, with a recovery of approximately 7%.     

Apical Surface Expression of Aspartic Protease Plasmepsin 4, a Potential Transmission-blocking Target of the Plasmodium Ookinete

To invade its definitive host, the mosquito, the malaria parasite must cross the midgut peritrophic matrix that is composed of chitin cross-linked by chitin-binding proteins and then develop into an oocyst on the midgut basal lamina. Previous evidence indicates that Plasmodium ookinete-secreted chitinase is important in midgut invasion. The mechanistic role of other ookinete-secreted enzymes in midgut invasion has not been previously examined. De novo mass spectrometry sequencing of a protein obtained by benzamidine affinity column of Plasmodium gallinaceum ookinete axenic culture supernatant demonstrated the presence of an ookinete-secreted plasmepsin, an aspartic protease previously only known to be present in the digestive vacuole of asexual stage malaria parasites. This plasmepsin, the ortholog of Plasmodium falciparum plasmepsin 4, was designated PgPM4. PgPM4 and PgCHT2 (the P. gallinaceum ortholog of P. falciparum chitinase PfCHT1) are both localized on the ookinete apical surface, and both are present in micronemes. Aspartic protease inhibitors (peptidomimetic and natural product), calpain inhibitors, and anti-PgPM4 monoclonal antibodies significantly reduced parasite infectivity for mosquitoes. These results suggest that plasmepsin 4, previously known only to function in the digestive vacuole of asexual blood stage Plasmodium, plays a role in how the ookinete interacts with the mosquito midgut interactions as it becomes an oocyst. These data are the first to delineate a role for an aspartic protease in mediating Plasmodium invasion of the mosquito and demonstrate the potential for plasmepsin 4 as a malaria transmission-blocking vaccine target.     

Exploration of the P3 region of PEXEL peptidomimetics leads to a potent inhibitor of the Plasmodium protease,plasmepsin V

The use of arginine isosteres is a known strategy to overcome poor membrane permeability commonly associated with peptides or peptidomimetics that possess this highly polar amino acid. Here, we apply this strategy to peptidomimetics that are potent inhibitors of the malarial protease, plasmepsin V, with the aim of enhancing their activity against Plasmodium parasites, and exploring the structure–activity relationship of the P₃ arginine within the S₃ pocket of plasmepsin V. Of the arginine isosteres trialled in the P₃ position, we discovered that canavanine was the ideal and that this peptidomimetic potently inhibits plasmepsin V, efficiently blocks protein export and inhibits parasite growth. Structure studies of the peptidomimetics bound to plasmepsin V provided insight into the structural basis for the enzyme activity observed in vitro and provides further evidence why plasmepsin V is highly sensitive to substrate modification.     

Multiple Amb a I allergens demonstrate specific reactivity with IgE and T cells from ragweed-allergic patients.

The relationship between the structure and abundance of an inhaled protein and its potential for causing an allergic response is unknown. This study analyzes Amb a I, a family of related proteins formerly known as Ag E, that comprise the major allergens of short ragweed (Ambrosia artemisiifolia). T cells isolated from ragweed allergic patients were shown to proliferate in response to purified Amb a I.1 protein from pollen in in vitro secondary cultures, demonstrating the presence of T cell stimulatory epitopes in Amb a I.1. Three recombinant forms of Amb a I (Amb a I.1, Amb a I.2, and Amb a I.3) obtained as cDNA derived from pollen mRNA were expressed in bacteria. All three recombinant forms were shown to be specifically recognized by pooled ragweed-allergic human IgE on immunoblots, confirming these gene products are important allergens. An examination of immunoblots probed with sera derived from allergic patients revealed a variation in IgE binding specificity. A minority of patients' IgE exclusively reacted with recombinant Amb a I.1, whereas most patients' IgE reacted with Amb a I.1 as well as Amb a I.2 and Amb a I.3 proteins. A detailed examination of the reactivity of T cells derived from 12 allergic patients to these recombinant Amb a I forms revealed that these allergens are all capable of stimulating T cell proliferation in in vitro assays. It is concluded that the allergic response to ragweed pollen in most allergic patients is composed of a reaction to multiple related Amb a I proteins at both the B and T cell levels.     

Alternate substrate inhibition of cholesterol esterase by thieno[2,3-d][1,3]oxazin-4-ones

In a kinetic study, the interaction of bovine pancreatic cholesterol esterase (CEase) with fused 1,3-oxazin-4-ones and 1,3-thiazin-4-ones was investigated, and the compounds were characterized as alternate substrate inhibitors. Inhibition assays were performed in the presence of sodium taurocholate with p-nitrophenyl butyrate as chromogenic substrate. Strong active site-directed inhibition was detected for 2-diethylaminothieno[2,3-d][1,3]oxazin-4-ones with a cycloaliphatic chain at positions 5,6. The most potent inhibitors, compounds 3 and 4, exhibited K(i) values of 0.58 and 1.86 microm, respectively. An exchange of the ring oxygen by sulfur and the removal of the cycloaliphatic moiety as well as the replacement of the thiophene ring by benzene led to a loss of inhibitory potency. CEase has the capability to catalyze the hydrolysis of representatives of fused 1,3-oxazin-4-ones as well as the highly stable 1,3-thiazin-4-ones by using an acylation-deacylation mechanism. Hydrolyses were performed in the presence of a high enzyme concentration, and products were identified spectrophotometrically and by means of high performance liquid chromatography. The kinetic parameters V(max)I and V(max)I/K(m)(I) for the CEase-catalyzed turnover were determined. The compounds whose enzyme-catalyzed hydrolysis followed first-order kinetics (K(m)(I) > 25 microm) failed to inhibit CEase. On the other hand, inhibitors 3 (initial concentration of 25 microm) and 4 (20 microm) were hydrolyzed by CEase under steady-state conditions in the first phase of the reaction. Rate-limiting deacylation was demonstrated in nucleophilic competition experiments with ethanol as acyl acceptor wherein the conversion of compound 3 was accelerated up to an ethanol concentration of 1.5 m. The characterization of these compounds (i.e. 3 and 4) as alternate substrate inhibitors is not only based on the verification of the CEase-catalyzed hydrolysis. It also rests upon the concurrence of corresponding K(i) values obtained in the inhibition assay compared with separately determined K(m)(I) values of their enzyme-catalyzed consumption, as could be predicted from the kinetic model used in this study.     

The role of the non-conserved residue at position 104 of class A beta-lactamases in susceptibility to mechanism-based inhibitors

The role of the non-conserved amino acid residue at position 104 of the class A beta-lactamases, which comprises a highly conserved sequence of amino acids at the active sites of these enzymes, in both the hydrolysis of beta-lactam substrates and inactivation by mechanism-based inhibitors was investigated. Site-directed mutagenesis was performed on the penPC gene encoding the Bacillus cereus 569/H beta-lactamase I to replace Asp104 with the corresponding Staphylococcus aureus PC1 residue Ala104. Kinetic data obtained with the purified Asp104Ala B. cereus 569/H beta-lactamase I was compared to that obtained from the wild-type B. cereus and S. aureus enzymes. Replacement of amino acid residue 104 had little effect on the Michaelis parameters for the hydrolysis of both S- and A-type penicillins. Relative to wild-type enzyme, the Asp104Ala beta-lactamase I had 2-fold higher Km values for benzylpenicillin and methicillin, but negligible difference in Km for ampicillin and oxacillin. However, kcat values were also slightly increased resulting in little change in catalytic efficiency, kcat/Km. In contrast, the Asp104Ala beta-lactamase I became more like the S. aureus enzyme in its response to the mechanism-based inhibitors clavulanic acid and 6-beta-(trifluoromethane sulfonyl)amido-penicillanic acid sulfone with respect to both response to the inhibitors and subsequent enzymatic properties. Based on the known three-dimensional structures of the Bacillus licheniformis 749/C, Escherichia coli TEM and S. aureus PC1 beta-lactamases, a model for the role of the non-conserved residue at position 104 in the process of inactivation by mechanism-based inhibitors is proposed.