Hydrolysis of thioester analogs by rat liver phospholipase A1

The hydrolysis of thioester containing phospholipids by rat liver plasmalemma phospholipase A1 was measured in a continuous spectrophotometric assay. In this assay thioester substrates were employed which, upon hydrolysis, liberated a free thiol which was reacted with 4,4'-dithiopyridine to yield the product 4-thiopyridone that absorbs at 324 nm. Thioester substrates, prepared by chemical synthesis, were used in phospholipid and Triton X-100 micelles for kinetic analysis carried out according to the method of Hendrickson and Dennis (Hendrickson, H.S., and Dennis, E.A. (1984) J. Biol. Chem. 259, 5734-5739). Vmax, Ks, and Km values obtained for various isomers and racemic mixtures of the synthetic thioester analogs are compared with corresponding oxyester substrates. Unnatural sn-1 isomers competitively inhibited the hydrolysis of natural sn-3 isomers of phosphatidylethanolamine and phosphatidic acid. Furthermore, the sn-1 isomer of phosphatidic acid was hydrolyzed by phospholipase A1, but with lower catalytic efficiency than the sn-3 isomer. The presence of a thioester at the sn-1 position did not change the Vmax significantly, as compared to the oxyester phospholipids. When two thioesters were present on the phospholipid molecule, the Vmax was decreased significantly. A convenient synthesis of 1-monothioester analogs of phospholipids is reported. The results presented show the usefulness of the spectrophotometric assay for measuring phospholipase A1 activity as well as the influence of racemic mixtures and thioesters on the hydrolytic rate.     

Amide bonds to the nitrogen atoms of cysteine and serine as "weak points" in the backbones of proteins

During the initial event in protein self-splicing, a peptide bond to the nitrogen atom of an internal cysteine or serine residue is usually cleaved by the side chain -SH or -OH group to yield a thioester or oxyester intermediate that undergoes further reactions. Self-splicing reactions also accompany the maturation of hedgehog signaling proteins, plant-type asparaginases, and pyruvoyl enzymes. It would be of interest to know whether peptide bonds that involve the nitrogen atoms of cysteine or serine are more susceptible to cleavage than peptide bonds to amino acids that lack reactive side chains. Extrapolations of the results of model reactions conducted at elevated temperatures indicate that the -SH group of N-acetylcysteine enhances the rate of its hydrolysis by a factor of 70, while the OH group of N-acetylserine enhances the rate of its hydrolysis 12-fold, compared with the rate of hydrolysis of N-acetylalanine in neutral solution at 25 °C. Several lines of evidence suggest that the rate-enhancing effects of these -SH and -OH side chains arise from their ability to act as intramolecular general acid-base catalysts for hydrolysis, rather than as nucleophilic catalysts. The protein environment within self-splicing proteins appears to redirect the actions of these side chains to nucleophilic attack, generating rate enhancements that approach the rate enhancements generated by conventional enzymes.     

The 3-hydroxy group and 4,5-trans double bond of sphingomyelin are essential for modulation of galactosylceramide transmembrane asymmetry

The structural features of SPM that control the transbilayer distribution of beta-GalCer in POPC vesicles were investigated by (13)C- and (31)P-NMR spectroscopy using lipid analogs that share physical similarities with GalCer or SPM. The SPM analogs included N-palmitoyl-4,5-dihydro-SPM, 3-deoxy-SPM, 1-alkyl-2-amidophosphatidylcholine, and dipalmitoylphosphatidylcholine, a popular model "raft lipid". The transbilayer distributions of the SPM analogs and SPM in POPC vesicles were similar by (31)P-NMR. To observe the dramatic change in GalCer transbilayer distribution that occurs when SPM is included in POPC vesicles, the 3-OH group, 4,5-trans double bond, and amide linkage all were required in SPM. However, inclusion of 2 and 10 mol % dihydroSPM in SPM/POPC (1:1) vesicles mitigated and completely abrogated the effect of SPM on the transbilayer distribution of GalCer. Despite sharing some structural features with GalCer and localizing preferentially to the inner leaflet of POPC vesicles, dimyristoylphosphatidylethanolamine did not undergo a change in transbilayer distribution when SPM was incorporated into the vesicles. The results support the hypothesis that specific interactions may be favored among select sphingolipids in curvature-stressed membranes and emphasize the potential importance of the SPM-dihydroSPM ratio in membrane fission and fusion processes associated with vesicle biogenesis and trafficking.     

Transbilayer distribution and movement of lysophosphatidylcholine in liposomal membranes

Single bilayer vesicles were prepared by sonication of 5 mol% 1-palmitoyl lysophosphatidylcholine and 95 mol% egg phosphatidylcholine. Incubation with lysophospholipase results in a fast hydrolysis of 80–90% of lysophosphatidylcholine. The remaining lysophosphatidylcholine is only very slowly hydrolysed. There results are interpreted as lysophosphatidylcholine being asymmetrically distributed over the two halves of the bilayer. The slow phase of lysophosphatidylcholine hydrolysis sets an upper limit to the rate of transbilayer movement of lysophosphatidylcholine. The half time of this process at 37° C is estimated to be about 100 h. Incorporation of cholesterol in the vesicles reduces the distributional asymmetry of lysophosphatidylcholine to the extent of an outside-inside ratio of 60 : 40. [¹⁴C]Lysophosphatidylcholine introduced into the outer monolayer of such vesicles by intervesicular transfer of lysophosphatidylcholine remains virtually completely available for hydrolysis by lysophospholipases, corroborating the interpretation that transbilayer movement of lysophosphatidylcholine in these vesicles is an extremely slow process.In handshaken liposomes consisting of 5 mol% 1-palmitoyl lysophosphatidylcholine and 95 mol% egg phosphatidylcholine 15–20% of lysophosphatidylcholine is readily available for exogenous lysophospholipase. This pool may represent lysophosphatidylcholine in the outer monolayer of the liposomes.     

Interfacial catalysis by phospholipase A2: the rate-limiting step for enzymatic turnover.

The kinetics of the phospholipase A2-catalyzed hydrolysis of bilayer vesicles and mixed micelles of several oxyglycero and thioglycero analogues of phospholipids have been studied. The results with vesicles show that, depending on the source of the enzyme, the rates of hydrolysis of the oxy-containing long-chain phosphatidylmethanols are 2.5- to 28-fold higher compared to the rates of hydrolysis of the analogous thio substrates. The oxygen to sulfur substitution does not significantly alter the affinities of the enzymes for the reaction products or calcium. Since it is unlikely that sulfur substitution changes the rate constants for the formation and dissociation of the enzyme-product complex by the same factor, the element effects seen in the rates of hydrolysis of the oxy- and thioester phospholipids in vesicles are primarily due to a change in the rate constant for the chemical step of the catalytic turnover cycle. For bovine pancreatic phospholipase A2, various mutants with lower catalytic activity were used to show that the value of the element effect does not increase in the mutants. These results establish that, for the pancreatic phospholipase A2, the element effect is fully expressed, and the chemical step is fully rate-limiting for both oxyglycero and thioglycero phospholipids in vesicles. It was found that the element effect decreases from 7 to 1 when long-chain phosphatidylmethanols are present in micelles of a neutral diluent. This result suggests that the chemical step is not rate-limiting during the hydrolysis of these mixed micelle substrates.     

Sphingomyelinase activity causes transbilayer lipid translocation in model and cell membranes

Ceramide is known to induce structural rearrangements in membrane bilayers, including the formation of ceramide-rich and -poor domains and the efflux of aqueous solutes. This report describes a novel effect of ceramide, namely the induction of transbilayer lipid movements. This effect was demonstrated in both model (large unilamellar vesicles) and cell (erythrocyte ghost) membranes in which ceramide generation took place in situ through the action of an externally added sphingomyelinase. Two different novel assays were developed to detect transbilayer lipid movement. One of the assays required the preparation of vesicles containing a ganglioside only in the outer monolayer and entrapped neuraminidase. Sphingomyelinase activity induced ganglioside hydrolysis under conditions in which no neuraminidase was released from the vesicles. The second assay involved the preparation of liposomes or erythrocyte ghosts labeled with a fluorescent energy donor in their inner leaflets. Sphingomyelin hydrolysis was accompanied by fluorescence energy transfer to an impermeable acceptor in the outer aqueous medium. Ceramide-induced transbilayer lipid movement is explained in terms of another well known property of ceramide, namely the facilitation of lamellar to non-lamellar lipid-phase transitions. Thus, sphingomyelinase generates ceramide on one side of the membrane; ceramide then induces the transient formation of non-lamellar structural intermediates, which cause the loss of lipid asymmetry in the bilayer, i.e. the transbilayer movement of ceramide together with other lipids. As direct targets for ceramide tend to be intracellular, these observations may be relevant to the mechanism of transmembrane signaling by means of the sphingomyelin pathway.     

Human complement proteins D, C2, and B. Active site mapping with peptide thioester substrates

The specificity and reactivity of complement serine proteases D, B, Bb, C2, and C2a were determined using a series of peptide thioester substrates. The rates of thioester hydrolysis were measured using assay mixtures containing the thiol reagent 4,4'-dithiodipyridine at pH 7.5. Each substrate contained a P1 arginine residue, and the effect of various groups and amino acids in the P2, P3, P4, and P5 positions was determined using kcat/Km values to compare reactivities. Among peptide thioesters corresponding to the activation site sequence in B, dipeptide thioesters containing a P2 lysine residue were the best substrates for D. Extending the chain to include a P3 or P4 amino acid resulted in loss of activity, and neither the tripeptide nor the tetrapeptide containing the cleavage sequence of B was hydrolyzed. Overall, D cleaved fewer substrates and was 2-3 orders of magnitude less reactive than C1s against some thioester substrates. C2 and fragment C2a had comparable reactivities and hydrolyzed peptides containing Leu-Ala-Arg and Leu-Gly-Arg, which have the same sequence as the cleavage sites of C3 and C5, respectively. The best substrates for C2 and C2a were Z-Gly-Leu-Ala-Arg-SBzl and Z-Leu-Gly-Leu-Ala-Arg-SBzl, respectively, where Bzl is benzyl. B was the least reactive among these complement enzymes. The best substrate for B was Z-Lys-Arg-SBzl with a kcat/Km value of 1370 M-1 s-1. The catalytic fragment of B, Bb, had higher activity toward these peptide thioester substrates. The best substrate for Bb was Z-Gly-Leu-Ala-Arg-SBzl with a kcat/Km similar to C2a and 10 times higher than the value for B. Both C2a and Bb were considerably more reactive against C3-like than C5-like substrates. Bovine trypsin hydrolyzed thioester substrates with kcat/Km approximately 10(3) higher than the complement enzymes. These thioester substrates for D, B, and C2 should be quite useful in kinetic and active site studies of the purified enzymes.     

Oligoglyceric acid synthesis by autocondensation of glyceroyl thioester

Summary The autocondensation of the glyceroyl thioesterS-glyceroyl-ethanethiol yielded oligoglyceric acid. The rates of autocondensation and hydrolysis of the thioester increased from pH 6.5 to pH 7.5 in 2,6-lutidine and imidazole buffers. Autocondensation and hydrolysis were much more rapid in imidazole buffers than in 2,6-lutidine buffers of the same pH. The efficiency of ester bond synthesis was about 20% for 40 mMS-glyceroylethanethiol in 2,6-lutidine and imidazole buffers near neutral pH. The size and yield of the oligoglyceric acid products increased when the concentration of the thioester was increased. The relationship of these results to prebiotic polymer synthesis is discussed.     

Hydrolysis of polyphosphoinositides by purified sheep seminal vesicle phospholipase C enzymes

Sheep seminal vesicles contain two immunologically distinct phospholipase C (PLC) enzymes that can hydrolyze phosphatidylinositol (PI) (Hofmann, S.L., and Majerus, P.W. (1982) J. Biol. Chem. 257, 6461-6469). One of these enzymes (PLC-I) has been purified to homogeneity; the second (PLC-II) has been purified 2600-fold from a crude extract of seminal vesicles. In the present study we have compared the ability of these purified enzymes to hydrolyze PI, phosphatidylinositol 4-phosphate (PI-4-P), and phosphatidylinositol 4,5-diphosphate (PI-4,5-P2). Using radiolabeled substrates in small unilamellar phospholipid vesicles of defined composition, the two enzymes were found to hydrolyze all three of the phosphoinositides. Hydrolysis of all three phosphoinositides by both enzymes was stimulated by Ca2+; however, in the presence of EGTA only the polyphosphoinositides were hydrolyzed. The two enzymes displayed substrate affinities in the order PI greater than PI-4-P greater than PI-4,5-P2, and maximum hydrolysis rates in the order PI-4,5-P2 greater than PI-4-P greater than PI. When present in the same vesicles, PI and the polyphosphoinositides competed for a limiting amount of either enzyme. Inclusion of phosphatidylcholine into vesicles containing the phosphoinositides resulted in greater inhibition of PI hydrolysis than polyphosphoinositide hydrolysis. When all three phosphoinositides were present in vesicles mimicking the cytoplasmic leaflet of cell membranes, there was preferential hydrolysis of the polyphosphoinositides over PI. We conclude that a single phospholipase C can account for the hydrolysis of all three phosphoinositides seen during agonist-induced stimulation of secretory cells. The cytoplasmic Ca2+ concentration and phospholipid composition of the membrane, however, may influence the relative rate of hydrolysis of the three phosphoinositides.     

Stoichiometry of reactions of alpha 2-macroglobulin with trypsin and chymotrypsin.

The stoichiometry of the individual steps, i.e. polypeptide chain cleavage, hydrolysis of the putative thioester bond and conformational change, of the reaction between alpha 2-macroglobulin and trypsin or chymotrypsin was analysed. The chain cleavage was monitored by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, the thioester hydrolysis by both a spectroscopic and a fluorimetric technique and the conformational change by tryptophan fluorescence. A stoichiometry of close to 2:1 was obtained for all reactions. This finding indicates that the alpha 2-macroglobulin half-molecule is an independent functional unit of the inhibitor, within which co-operativity between the two subunits may occur.     

Studies on chemical and enzymatic synthesis of maleyl-CoA

In the course of the enzymatic reaction of acetoacetyl-CoA with maleate, catalyzed by CoA transferase, a transient appearance of free CoA-SH occurred. Subsequently, both free CoA and acyl-CoA decreased with time, indicating the formation of an unusual CoA derivative resistant to alkaline hydrolysis. During the chemical reaction of CoA-SH with maleic anhydride, the SH groups of CoA disappeared quickly, but not more than 30% could be accounted for as thioester. The product is unstable at neutrality and is hydrolyzed by nitroprusside reagent. Another product having an acyl bond of low reactivity, which reacts with hydroxylamine and does not undergo ammonolysis, but is susceptible to alkaline hydrolysis, slowly accumulated, accounting for about 25% of the CoA that disappeared. The main product appears to be formed by the addition of CoA-SH to the double bond of maleic anhydride. Column chromatography of the products of the chemical and the enzymatic reaction revealed two products, designated at X1 and X2, showing absorbance of the adenine moiety of CoA, containing 14C-labeled maleate and no free SH groups. Alkaline hydrolysis of X1 resulted in the recovery of CoA and in an increase of X2. The results are interpreted as indicating that maleyl-CoA readily hydrolyzes and reacts spontaneously with the SH group of free CoA to form an addition compound. The thioester of this product slowly hydrolyzes to give rise to the final product, which appears to be the thioether, a stable and metabolically inert compound.     

The Effect of 2-Ketobutyrate on Mitochondrial Substrate-Level Phosphorylation

Neurochemical Research - The reaction catalyzed by succinate-CoA ligase in the mitochondrial matrix yields a high-energy phosphate when operating towards hydrolysis of the thioester bond of...     

Differential Ubiquitin Binding by the Acidic Loops of Ube2g1 and Ube2r1 Enzymes Distinguishes Their Lys-48-ubiquitylation Activities

The ubiquitin E2 enzymes, Ube2g1 and Ube2r1, are able to synthesize Lys-48-linked polyubiquitins without an E3 ligase but how that is accomplished has been unclear. Although both E2s contain essential acidic loops, only Ube2r1 requires an additional C-terminal extension (184–196) for efficient Lys-48-ubiquitylation activity. The presence of Tyr-102 and Tyr-104 in the Ube2g1 acidic loop enhanced both ubiquitin binding and Lys-48-ubiquitylation and distinguished Ube2g1 from the otherwise similar truncated Ube2r1^1–183 (Ube2r1C). Replacement of Gln-105–Ser-106–Gly-107 in the acidic loop of Ube2r1C (Ube2r1C^YGY) by the corresponding residues from Ube2g1 (Tyr-102–Gly-103–Tyr-104) increased Lys-48-ubiquitylation activity and ubiquitin binding. Two E2∼UB thioester mimics (oxyester and disulfide) were prepared to characterize the ubiquitin binding activity of the acidic loop. The oxyester but not the disulfide derivative was found to be a functional equivalent of the E2∼UB thioester. The ubiquitin moiety of the Ube2r1C^C93S-[¹⁵N]UB^K48R oxyester displayed two-state conformational exchange, whereas the Ube2r1C^C93S/YGY-[¹⁵N]UB^K48R oxyester showed predominantly one state. Together with NMR studies that compared UB^K48R oxyesters of the wild-type and the acidic loop mutant (Y102G/Y104G) forms of Ube2g1, in vitro ubiquitylation assays with various mutation forms of the E2s revealed how the intramolecular interaction between the acidic loop and the attached donor ubiquitin regulates Lys-48-ubiquitylation activity.     

Inhibitors can activate proteases to catalyze the synthesis and hydrolysis of peptides

Competitive inhibitors can activate proteases (papain, trypsin, and cathepsin S) to catalyze the synthesis of peptide bonds and accelerate the hydrolysis of poor substrates (from 1 to 99%). Reaction mixtures contained intermediate molecules that were formed by the coupling of the inhibitor with the poor substrate. This and other findings suggest the following chain of events. Part of the binding energy of formation of the enzyme-inhibitor complex was used to activate the inhibitor, i.e., to form acyl-enzyme species with a high-energy bond (e.g., a thioester bond in the case of papain) required for coupling the inhibitor with the substrate to form the intermediate molecule. The latter was subjected to successive reactions which led to a stepwise degradation of the substrate, as well as to the regeneration of the inhibitor. One mole of the inhibitor could catalyze rapid hydrolysis of at least 53 mol of substrate. The intermediate molecules were the species undergoing rapid hydrolysis. Therefore, 1 mol of inhibitor was involved in the synthesis of 53 mol of intermediate molecules; i.e., the inhibitor functioned as a cofactor that catalyzed the synthesis of peptides. Thus, the binding energy of formation of the enzyme-inhibitor complex can be utilized to catalyze the synthesis of peptide bonds in the absence of an exogenous energy source (e.g., ATP).     

Peptide thioesters and 4-nitroanilides as substrates for porcine pancreatic kallikrein

A series of 14 4-nitroanilide substrates and 17 thioester substrates have been used to measure kinetic constants with porcine pancreatic kallikrein. All of the substrates have a P1 arginine residue. The 4-nitroanilide substrates consist of seven P2-glycine and seven P2-phenylalanine tripeptides. As expected from previous results, the phenylalanine series substrates were generally 100-fold 'better' than those in the glycine series. The S3 subsite was found to 'prefer' lysine or phenylalanine, whereas glutamic acid in this position was distinctly unfavourable. The thioester substrates consisted of various thioester derivatives of arginine as well as 12 dipeptides. These substrates exhibited kcat./Km values generally 1000 times higher than the P2-phenylalanine 4-nitroanilides. With the thioesters, a P2 phenylalanine or tryptophan residue yielded the best substrates, but some of the simple derivatives of arginine were nearly as good. A comparison of the kinetic constants of the thioester substrates between the porcine enzyme and human plasma kallikrein provides further evidence that these enzymes have a similar preference for bulky P2 residues, but otherwise are quite different enzymes. The thioester substrates are nearly as reactive as oxygen ester substrates such as acetylphenylalanylarginine methyl ester for the porcine enzyme [Levison & Tomalin (1982) Biochem. J. 203, 299-302; Fiedler (1983) Adv. Exp. Med. Biol. 156A, 263-274], and owing to the greater ease in assaying with the thioesters, they should find use in routine assays for the glandular kallikreins.     

Glyoxalase II from Zea mays: properties and inhibition study of the enzyme purified by use of a new affinity ligand

The synthesis of N-(p-nitrocarbobenzoxy)glutathione (N-pNCBG) is reported. N-pNCBG and glutathione (GSH) were coupled to Affi-gel 10 by a thioester linkage and resulted in very effective bound ligands for a fast purification of glyoxalase II from corn. The S-(N-pNCBG)-affinity column showed a glyoxalase II binding capacity of up to 2-fold higher than that of the glutathione-affinity column. A single form of glyoxalase II was evidenced by PAGE in both crude extracts and in the affinity purified enzyme. A 45% recovery of glyoxalase II activity (purification, approx. 433-fold) was obtained for both matrices by a single chromatography. The purified glyoxalase is an acidic protein (pI 4.5) of about 26,000 relative molecular mass. Substrate studies for the corn glyoxalase II show, among possible substrates tested, that S-D-lactyl-glutathione is the preferred substrate. An inhibition study was performed with methyl-, propyl-, hexyl-, p-nitrobenzyl-, p-chlorophenacyl-, carbobenzoxy-, and p-nitrocarbobenzoxy-S-glutathione. Methyl-S-glutathione did not inhibit corn glyoxalase II; the others were found to be linear competitive inhibitors. The derivatives containing a thioether bond are weaker inhibitors than those containing a thioester bond or a carbonyl group. p-Nitrobenzyl-S-glutathione is the weakest inhibitor; the carbobenzoxy-S-derivatives are stronger inhibitors than the p-chlorophenacyl S-derivative.     

The Curvature and cholesterol content of phospholipid bilayers alter the transbilayer distribution of specific molecular species of phosphatidylethanolamine

The curvature, cholesterol content,and transbilayer distribution of phospholipids significantly influence the functional properties of cellular membranes, yet little is known of how these parameters interact. In this study, the transbilayer distribution of phosphatidylethanolamine (PE) is determined in vesicles with large (98 nm) and small (19 nm)radii of curvature and with different proportions of PE, phosphatidylcholine, and cholesterol. It was found that the mean diameters of both types of vesicles were not influenced by their lipid composition, and that the amino-reactive compound 2,4,6-trinitrobenzenesulphonic acid (TNBS) was unable to cross the bilayer of either type of vesicle. When large vesicles were treated with TNBS, ~40% of the total membrane PE was derivatized; in the small vesicles 55% reacted. These values are interpreted as representing the percentage of total membrane PE residing in the outer leaflet of the vesicle bilayer. The large vesicles likely contained ~20% of the total membrane lipid as internal membranes. Therefore, in both types of vesicles, PE as a phospholipid class was randomly distributed between the inner and outer leaflets ofthe bilayer. The proportion oftotal PE residing in the outer leaflet was unaffected by changes in either the cholesterol orPE content of the vesicles. However, the transbilayer distributions of individual molecular species of PE were not random, and were significantly influenced by radius of curvature, membrane cholesterol content, or both. For example, palmitate and docosahexaenoate-containing species of PE were preferentially located in the outer leaflet of the bilayer. Membrane cholesterol content affected the transbilayer distributions of stearate-, oleate-, and linoleate-containing species. The transbilayer distributions ofpalmitate-, docosahexaenoate-, and stearate-containing species were significantly influenced by membrane curvature, but only in the presence of high levels of cholesterol. Thus, differences in membrane curvature and cholesterol content alter the array of PE molecules present on the surfaces of phospholipid bilayers. In cells and organelles, these differences could have profound effects on a number of critical membrane functions and processes.     

Evidence for frequent OspC gene transfer between Borrelia valaisiana sp. nov. and other Lyme disease spirochetes

IMP-1 metallo-beta-lactamase is a transferable carbapenem-hydrolyzing enzyme found in some clinical isolates of Pseudomonas aeruginosa, Serratia marcescens and Klebsiella pneumoniae. Bacteria that express IMP-1 show significantly reduced sensitivity to carbapenems and other beta-lactam antibiotics. A series of thioester derivatives has been shown to competitively inhibit purified IMP-1. As substrates for IMP-1, the thioesters yielded thiol hydrolysis products which themselves were reversible competitive inhibitors. The thioesters also increased sensitivity to the carbapenem L-742,728 in an IMP-1-producing laboratory stain of Escherichia coli, but will need further modification to improve their activity in less permeable organisms such as Pseudomonas and Serratia. Nonetheless, the thioester IMP-1 inhibitors offer an encouraging start to overcoming metallo-beta-lactamase-mediated resistance in bacteria.