The kinetics of hydrolysis of some extended N-aminoacyl-L-arginine methyl esters by porcine pancreatic kallikrein. A comparison with human plasma Kallikrein.

The effects of subsite interactions in the S2-S4 region [Schechter & Berger (1967) Biochem. Biophys. Res. Commun. 27, 157-162] of porcine pancreatic kallikrein (EC 3.4.21.8) on its catalytic efficiency have been investigated. Kinetic constants (Kcat, Km) have been determined for a series of seven extended N-aminoacyl-L-arginine methyl esters whose sequence is based on either the C-terminal sequence of kallidin (-Pro-Phe-Arg) or (-Gly-)nArg. With these substrates it has been found that neither acylation nor deacylation of the enzyme is rate-limiting. Values of Kcat. range from 21.5 to 2320s-1, indicating that there are interactions with different residues in the N-aminoacyl chain and enzyme subsites in the S2-S4 region. It is shown that possible hydrogen-bonded interactions with the enzyme in the S3-S4 region have a significant effect on catalysis. The presence of L-phenylalanine at P2 has a very large effect on both Kcat, and Km, giving a greatly enhanced catalytic efficiency. Substrates with L-proline at P3 also have a marked effect, but in this case the overall effect is one of lowered catalytic efficiency. By comparison with the results of a similar study with human plasma kallikrein I (EC 3.4.21.8), it has been possible to demonstrate that there are considerable differences in kinetic behaviour between the two enzymes. These are related to relative differences in the rates of acylation and deacylation with ester substrates and also the roles of subsites S2 and S3 of the two enzymes.     

Catalysis by human leukocyte elastase: III. Steady-state kinetics for the hydrolysis of p-nitrophenyl esters

Steady-state kinetic parameters were determined at pH 7.4 and 25 degrees C for the human leukocyte elastase-catalyzed hydrolysis of several N-carbobenzoxy-L-amino acid p-nitrophenyl esters. The substrate specificity for these esters was quite broad, and included the Gly, Phe, and Tyr derivatives. Together with reports of a much narrower P-1 specificity for peptide-based substrates, these results suggest that interactions remote from the scissle bond between enzyme and substrate regulate primary specificity. Also, it was found that kc and kc/Km did not exhibit the same dependence on substrate structure. This is interpreted to suggest that there are significant differences in P-1 specificity between acylation and deacylation for leukocyte elastase-catalyzed reactions.     

S(1)' and S(2)' subsite specificities of human plasma kallikrein and tissue kallikrein 1 for the hydrolysis of peptides derived from the bradykinin domain of human kininogen

The S(1)' and S(2)' subsite specificities of human tissue kallikrein 1 (KLK1) and human plasma kallikrein (HPK) were examined with the peptide series Abz-GFSPFRXSRIQ-EDDnp and Abz-GFSPFRSXRIQ-EDDnp [X=natural amino acids or S(PO(3)H(2))]. KLK1 efficiently hydrolyzed most of the peptides except those containing negatively charged amino acids at P(1)' and P(2)' positions. Abz-GFSPFRSSRIQ-EDDnp, as in human kininogen, is the best substrate for KLK1 and exclusively cleaved the R-S bond. All other peptides were cleaved also at the F-R bond. The synthetic human kininogen segment Abz-MISLMKRPPGFSPFRS(390)S(391)RI-NH(2) was hydrolyzed by KLK1 first at R-S and then at M-K bonds, releasing Lys-bradykinin. In the S(390) and S(391) phosphorylated analogs, this order of hydrolysis was inverted due to the higher resistance of the R-S bond. Abz-MISLMKRPPG-FSPFRSS(PO(3)H(2))(391)RI-NH(2) was hydrolyzed by KLK1 at M-K and mainly at the F-R bond, releasing des-(Arg(9))-Lys-Bk which is a B1 receptor agonist. HPK cleaved all the peptides at R and showed restricted specificity for S in the S(1)' subsite, with lower specificity for the S(2)' subsite. Abz-MISLMKRPPGFSPFRSSRI-NH(2) was efficiently hydrolyzed by HPK under bradykinin release, while the analogs containing S(PO(3)H(2)) were poorly hydrolyzed. In conclusion, S(1)' and S(2)' subsite specificities of KLK1 and HPK showed peculiarities that were observed with substrates containing the amino acid sequence of human kininogen.     

Leishmania amazonensis: uptake and hydrolysis of 3H-amino acid methyl esters by isolated amastigotes

Intracellular and isolated amastigotes of Leishmania amazonensis can be destroyed by L-amino acid methyl esters known to disrupt mammalian lysosomes. To evaluate the mechanism(s) involved in the leishmanicidal activity, we examined the uptake and hydrolysis of tritiated esters by isolated amastigotes. After incubation with the labeled compounds, parasites were recovered, were washed on filters, and their radioactivity was determined. Alternatively, amastigotes were separated from the medium by centrifugation through oil, and the radioactivity associated with free or esterified amino acids was measured after thin-layer chromatography. The results showed that the methyl esters of Trp, Leu, and Met, which are leishmanicidal, accumulated in and were rapidly hydrolysed by the amastigotes. [3H]Leu derived from [3H]Leu-OMe remained associated with the amastigotes even after a 1-hr chase in label-free medium, but the ester species was rapidly lost upon washing of the parasites. In contrast, the esters of Ile and Ala, which are not leishmanicidal, were only slowly hydrolysed, and most of the radioactivity was lost upon washing. We have previously shown that certain amino acid esters and weak bases protect Leishmania from damage by leucine methyl ester (Leu-OMe). In the present experiments, these compounds reduced, in concentration-dependent fashion, the hydrolysis of [3H]Leu-OMe and the accumulation of [3H]Leu in the amastigotes. Overall, the results indicate that, as in lysosomal disruption, leishmanicidal activity is associated with ester hydrolysis and amino acid accumulation in the parasites. The nature and location of the parasite esterolytic enzymes requires additional investigation.     

The use of microwave oven for the rapid hydrolysis of bile acid methyl esters.

An efficient and convenient procedure for the hydrolysis of bile acid methyl esters is described. This is achieved by the addition of aqueous lithium hydroxide in methanol/dioxane/tetrahydrofuran (or dimethylformamide) in the microwave oven. Under these conditions the formates as well as the acetate derivatives prepared under microwave irradiation conditions were also hydrolyzed, and the desired bile acids were isolated in 86-94% yield. All these reactions were completed in the microwave oven within 45-60 s.     

The specificity of the S1 and S2 subsites of elastase

Esters of tetrapeptides of the general formula ethoxycarbonyl-prolyl-alanyl-X-Y where either X or Y was an alanine residue were synthesised and their cleavage by elastase studied. It was found that variation of the alcohol moiety between methyl, cyclohexyl and nitrophenyl residues had no effect on the catalytic rate constant for cleavage of ethoxycarbonyl-prolyl-dialanyl-alanine esters demonstrating that acylation is much faster than deacylation for this system and also that non-productive binding is not kinetically significant. The effect of changing the amino acid residue in position X was small compared with that of change in position Y. The presence of valine and serine residues in position Y produced the highest specificity constant but the highest catalytic rate constant was found for a leucine residue in this position. The results are discussed in terms of the binding of the substrate to the enzyme.     

Plasticity of extended subsites facilitates divergent substrate recognition by Kex2 and furin

Yeast Kex2 and human furin are subtilisin-related proprotein convertases that function in the late secretory pathway and exhibit similar though distinguishable patterns of substrate recognition. Although both enzymes prefer Arg at P(1) and basic residues at P(2), the two differ in recognition of P(4) and P(6) residues. To probe P(4) and P(6) recognition by Kex2p, furin-like substitutions were made in the putative S(4) and S(6) subsites of Kex2. T252D and Q283E mutations were introduced to increase the preference for Arg at P(4) and P(6), respectively. Glu(255) was replaced with Ile to limit recognition of P(4) Arg. The effects of putative S(4) and S(6) mutations were determined by examining the cleavage by purified mutant enzymes of a series of fluorogenic substrates with systematic changes in P(4) and/or P(6). Whereas wild Kex2 exhibited little preference type for Arg at P(6), the T252D mutant and T252D/Q283E double mutant exhibited clear interactions with P(6) Arg. Moreover, the T252D and T252D/Q283E substitutions altered the influence of the P(6) residue on P(4) recognition. We infer that cross-talk between S(4) and S(6), not seen in furin, allows wild type and mutant forms of Kex2 to adapt their subsites for altered modes of recognition. This apparent plasticity may allow the subsites to rearrange their local environment to interact with different substrates in a productive manner. E255I-Kex2 exhibited significantly decreased recognition of P(4) Arg in a tetrapeptide substrate with Lys at P(1), although the general pattern of selectivity for aliphatic residues at P(4) remained unchanged.     

GTP binding and hydrolysis kinetics of human septin 2

Septins are a family of conserved proteins that are essential for cytokinesis in a wide range of organisms including fungi, Drosophila and mammals. In budding yeast, where they were first discovered, they are thought to form a filamentous ring at the bridge between the mother and bud cells. What regulates the assembly and function of septins, however, has remained obscure. All septins share a highly conserved domain related to those found in small GTPases, and septins have been shown to bind and hydrolyze GTP, although the properties of this domain and the relationship between polymerization and GTP binding/hydrolysis is unclear. Here we show that human septin 2 is phosphorylated in vivo at Ser218 by casein kinase II. In addition, we show that recombinant septin 2 binds guanine nucleotides with a Kd of 0.28 microm for GTPgammaS and 1.75 microm for GDP. It has a slow exchange rate of 7 x 10(-5) s(-1) for GTPgammaS and 5 x 10(-4) s(-1) for GDP, and an apparent kcat value of 2.7 x 10(-4) s(-1), similar to those of the Ras superfamily of GTPases. Interestingly, the nucleotide binding affinity appears to be altered by phosphorylation at Ser218. Finally, we show that a single septin protein can form homotypic filaments in vitro, whether bound to GDP or GTP.     

Novel non-peptidic vinylsulfones targeting the S2 and S3 subsites of parasite cysteine proteases

We describe here the identification of non-peptidic vinylsulfones that inhibit parasite cysteine proteases in vitro and inhibit the growth of Trypanosoma brucei brucei parasites in culture. A high resolution (1.75 Å) co-crystal structure of 8a bound to cruzain reveals how the non-peptidic P2/P3 moiety in such analogs bind the S2 and S3 subsites of the protease, effectively recapitulating important binding interactions present in more traditional peptide-based protease inhibitors and natural substrates.     

The pH dependence of the hydrolysis of chromogenic substrates of the type, Lys-Pro-Xaa-Yaa-Phe-(NO2)Phe-Arg-Leu, by selected aspartic proteinases: evidence for specific interactions in subsites S3 and S2

Variation in the kinetic parameters, kcat and Km, with pH has been used to obtain evidence for significant acid-dissociation processes in the hydrolysis of octapeptide substrates by three aspartic proteinases. These substrates are all cleaved at the peptide bond between a Phe (P1) and a p-nitroPhe (P1') residue resulting in a shift in absorbance at 300 nm that facilitates kinetic measurements. The substrates differ in the amino-acid residues present in the P3 and the P2 positions. Porcine pepsin, calf chymosin, and the aspartic proteinase from Endothia parasitica all show pH dependencies that imply that favorable or unfavorable interactions can occur with the S3 or S2 areas of the enzyme-active site. Examination of the crystallographically determined structure of the E. parasitica proteinase and consideration of the amino-acid sequence differences between the three enzymes suggests that the origin of the pH effects arises from favorable interactions between Glu-13 (COO-) of pig pepsin and Thr (OH) or His (ImH+) in P3 of a substrate. Similarly, Lys-220 (NH3+) of chymosin and a Glu (COO-) in P2 of a substrate may produce a favorable interaction and Asp-77 (COO-) of E. parasitica proteinase and a Glu (COO-) in P2 of a substrate may produce an unfavorable interaction. These results lead to possible explanations for subtle specificity differences within a family of homologous enzymes, and suggest loci for study by site-directed mutagenesis.     

The hydrolysis of cholesterol esters in plasma lipoproteins by hormone-sensitive cholesterol esterase from adipose tissue.

Adipose tissue contains a high level of neutral esterase active against emulsions of cholesteryl oleate. The present studies show that this enzyme can also effectively hydrolyze the cholesterol esters in native rat plasma high density lipoproteins (HDL) and low density lipoproteins (LDL). The hydrolysis of lipoprotein cholesterol esters by a pH 5.2 isoelectric precipitate fraction from the freshly prepared 100,000 X g supernatant of chicken adipose tissue was low, but increased more than 50-fold on activation with cyclic AMP-dependent protein kinase. Rat adipose tissue homogenates were also very active against lipoprotein cholesterol esters, hydrolyzing as much as 60% of the total labeled cholesterol ester in HDL or LDL in 1 h. Activity was optimal at pH 7 and very low at pH 4. No protease activity was detected at pH 7 and, since assays were done in 2 mM EDTA, phospholipase A activity was presumably negligible. The results show that hormone-sensitive cholesterol esterase of adipose tissue has ready access to the neutral lipid core of plasma lipoproteins, either because the enzyme penetrates the polar shell or because the cholesterol ester in the core is exposed, at least intermittently, to allow enzyme-substrate complex formation. Whether or not this enzyme activity plays a role in lipoprotein degradation by adipose tissue remains to be determined.     

Papain-catalysed hydrolysis of some hippuric esters. A new mechanism for papain-catalysed hydrolyses

1. The Michaelis–Menten parameters for the papain-catalysed hydrolysis of a number of alkyl, aryl and alkyl-thiol esters of hippuric acid have been determined. 2. For all the aryl esters and most of the alkyl esters studied, the catalytic constant, k₀, is 2–3sec.⁻¹ and most probably represents deacylation of the common intermediate, hippuryl-papain. 3. Two alkyl esters and hippurylamide, however, have catalytic rate constants, k₀, less than 2–3sec.⁻¹. It is possible to interpret all the available kinetic data in terms of a three-step mechanism in which an enzyme–substrate complex is first formed, followed by acylation of the enzyme through an essential thiol group, followed by deacylation of the acyl-enzyme. 4. The logarithm of the ratio of the Michaelis–Menten parameters, which reflect the acylation rate constant, for four aryl esters of hippuric acid studied give a linear Hammett plot against the substituent constant, σ. Arguments are presented that indicate acid as well as nucleophilic catalysis in the acylation process and that the most likely proton donor is an imidazolium ion. 5. It is suggested that this imidazolium ion is part of the same histidine residue that has been tentatively implicated in the deacylation process (Lowe & Williams, 1965b). 6. A new mechanism is proposed for the papain-catalysed hydrolysis of N-acyl-α-amino acid derivatives.     

Comparative study of hydrolysis of methyl esters of N-arylsulfonyl-valyl-arginine by thrombin and trypsin]

The esterase action of thrombin and trypsin on N-arylsulfonyl-valyl-arginine methyl esters was studied. The values of Km and kcat under steady-state conditions at pH 8,5 were determined. It was shown that the nature of the arylsulfonyl group does not affect the kinetic parameters of the reactions under study. The Michaelis constants of the thrombin-catalyzed reactions appeared to be one order of magnitude lower than the Km values of the corresponding TAME analogs.     

Proteases as catalysts of enantioselective α-amino ester hydrolysis: 3. Pronase-catalysed hydrolysis of methyl esters of leucine,phenylalanine and valine

An investigation of the hydrolysis of various substrates (l-Trp-OMe, l-Phe-OMe, l-Leu-OMe and l-Val-OMe) by pronase has shown that the highest activity is displayed with l-Leu-OMe substrate. Addition of Ca²⁺ ions significantly enhances the rate of hydrolysis of l-Leu-OMe without affecting the hydrolysis of d-Leu-OMe. Thus, the enantioselectivity of the process is improved.     

Various properties and kinetics of interaction of high and low molecular weight human kininogens with human plasma kallikrein

A relatively simple procedure for isolation and purification of human blood plasma kallikrein (HPK) by QAE-Sephadex A-50 SP-Sephadex C-50 and affinity chromatography on Sepharose 4B with immobilized soybean trypsin inhibitor with the activity yield of about 40% has been developed. The method allows for simultaneous isolation of low (LMW) and high molecular weight (HMW) kininogens from the same HPK sample. HPK preparations are homogeneous upon 7.5% polyacrylamide gel electrophoresis in the presence of 0.1% SDS; its Mr is 90,000. After treatment with beta-mercaptoethanol, HPK dissociates into two fragments with Mr of 43,000 and 37,000. HPK preparations have high specific activities of esterase (31 microM/min), amidase (78 microM/min), and kininogenase (420 micrograms equiv. bradikinin/min). The high degree of protein purification was demonstrated by titration of active centers with 4-methylumbelliferylguanidine benzoate. The values of equilibrium dissociation constants for the HPK complex with aprotinin (Ki) equal to 1 X 10(-8) M (ethyl ester of N-alpha-benzoyl-L-arginine) and 1,5 X 10(-9) M (HMW) were determined. The kinetics of HPK-induced liberation of bradikinin from purified preparations of HMW and LMW was studied. The kinetic parameters (Km, kcat and kcat/Km) of this reaction suggest a high affinity of HPK for HMW, but not for LMW. LMW does not compete with HMW for the enzyme active center. It is assumed that LMW is not a physiological substrate for HPK.     

Bacterial mutagenicity of some methyl 2-cyanoacrylates and methyl 2-cyano-3-phenylacrylates

The mutagenic potential of three alkyl 2-cyanoacrylate adhesives, three commercial alkyl 2-cyanoacrylate adhesives and three methyl 2-cyano-3-phenylacrylates, was assessed using the Salmonella/microsome mutagenicity assay. Compounds were tested with and without Aroclor 1254-induced rat-liver homogenate (S9 mix). The methyl 2-cyanoacrylate adhesives were mutagenic in the standard plate test with S. typhimurium strain TA100 with and without S9 activation. Methyl 2-cyano-3-(2-bromophenyl)acrylate revealed a direct mutagenic action to S. typhimurium strain TA1535. The compounds most toxic towards the bacterium S. typhimurium, were the methyl 2-cyanoacrylate adhesives (greater than 500 micrograms/plate). All alkyl 2-cyanoacrylate adhesives were tested in a modified spot test for volatile compounds with tester strain TA100. Mutagenic and toxic effects were observed with the three methyl 2-cyanoacrylate adhesives. It can be concluded from the results that the bacterial toxicity and mutagenicity of methyl 2-cyanoacrylate adhesives may be due to the methyl 2-cyanoacrylate monomer.     

Kinetic and modeling studies of S3-S3' subsites of HIV proteinases.

Kinetic analysis and modeling studies of HIV-1 and HIV-2 proteinases were carried out using the oligopeptide substrate [formula: see text] and its analogs containing single amino acid substitutions in P3-P3' positions. The two proteinases acted similarly on the substrates except those having certain hydrophobic amino acids at P2, P1, P2', and P3' positions (Ala, Leu, Met, Phe). Various amino acids seemed to be acceptable at P3 and P3' positions, while the P2 and P2' positions seemed to be more restrictive. Polar uncharged residues resulted in relatively good binding at P3 and P2 positions, while at P2' and P3' positions they gave very high Km values, indicating substantial differences in the respective S and S' subsites of the enzyme. Lys prevented substrate hydrolysis at any of the P2-P2' positions. The large differences for subsite preference at P2 and P2' positions seem to be at least partially due to the different internal interactions of P2 residue with P1', and P2' residue with P1. As expected on the basis of amino acid frequency in the naturally occurring cleavage sites, hydrophobic residues at P1 position resulted in cleavable peptides, while polar and beta-branched amino acids prevented hydrolysis. On the other hand, changing the P1' Pro to other amino acids prevented substrate hydrolysis, even if the substituted amino acid had produced a good substrate in other oligopeptides representing naturally occurring cleavage sites. The results suggest that the subsite specificity of the HIV proteinases may strongly depend on the sequence context of the substrate.