Catalytic Serine Residue Research Articles

Cloning and expression of a cDNA encoding a hepatic microsomal lipase that mobilizes stored triacylglycerol

A microsomal triacylglycerol hydrolase (TGH) was recently purified from porcine liver [Lehner and Verger (1997) Biochemistry 36, 1861-1868]. To gain further insight into the function of TGH, we have cloned a cDNA encoding TGH from a rat liver cDNA library and generated McArdle RH7777 rat hepatoma cell lines that stably express the rat TGH. The putative protein derived from the cDNA sequence contains a cleavable signal sequence and a catalytic site serine residue present within a pentapeptide motif (GXSXG) that is conserved in all known lipases. TGH-transfected cells showed a 2-fold increase, compared with control cells, in the rate of depletion of prelabelled triacylglycerol stores. Thus, TGH is capable of hydrolysis of stored triacylglycerol. In contrast, the rate of turnover of labelled phosphatidylcholine was similar in both the vector- and TGH-transfected cells. Studies in TGH-transfected cells demonstrated that utilization of intracellular triacylglycerol pools for secretion was approx. 30% higher than in vector-transfected cells. Whereas phosphatidylcholine secretion was essentially the same in control and TGH-transfected cells, TGH-transfected cells also secreted an approx. 25% greater mass of triacylglycerol into the medium and had increased levels of apolipoprotein B100 in the very-low-density lipoprotein density range compared with control cells. The results suggest that the microsomal TGH actively participates in the mobilization of cytoplasmic triacylglycerol stores, some of which can be used for lipoprotein assembly.

Cytoskeletal effects induced by pet, the serine protease enterotoxin of enteroaggregative Escherichia coli.

We have previously described enteroaggregative Escherichia coli (EAEC) strains that induce cytotoxic effects on T84 cells, ligated rat ileal loops, and human intestine in culture. Such strains secrete a 104-kDa protein termed Pet (for plasmid-encoded toxin). We have also shown previously that the Pet toxin induces rises in short-circuit current and decreases the electrical resistance in rat jejunum mounted in an Ussing chamber. The nucleotide sequence of the pet gene revealed that Pet is a member of the autotransporter class of secreted proteins. Here we show that a concentrated supernatant of E. coli HB101 harboring the minimal pet clone pCEFN1 induces temperature-, time- and dose-dependent cytopathic effects on HEp-2 cells and HT29 C1 cells in culture. The effects were characterized by release of the cellular focal contacts from the glass substratum, followed by complete rounding of the cells and detachment from the glass. Staining of the Pet-treated cells with Live/Dead viability stain revealed that >90% of rounded cells were viable. Pet-intoxicated HEp-2 and HT29 cells stained with fluorescein-labeled phalloidin revealed contraction of the cytoskeleton and loss of actin stress fibers. However, the effects of Pet were not inhibited by cytoskeleton-altering drugs, including colchicine, taxol, cytochalasin D, and phallicidin. The Pet protein induced proteolysis in zymogram gels, and preincubation with the serine protease inhibitor phenylmethylsulfonyl fluoride resulted in complete abrogation of Pet cytopathic effects. We introduced a mutation in a predicted catalytic serine residue and found that the mutant (Pet S260I) was deficient in protease activity and did not produce cytopathic effects, cytoskeletal damage, or enterotoxic effects in Ussing chambers. These data suggest that Pet is a cytoskeleton-altering toxin and that its protease activity is involved in each of the observed phenotypes.

Molecular cloning of neuropathy target esterase (NTE)

Covalent modification of NTE, a neuronal protein with serine esterase activity, by certain organophosphates (OP) initiates degeneration of long axons in the peripheral and central nervous system. Simple inhibition of NTE esterase activity does not initiate neuropathy; the latter requires aging of the OP bound to the catalytic serine residue so that a negatively-charged species is left attached to the active site. This may indicate that a non-esterase function of NTE is important for axonal maintenance. We have recently cloned NTE and shown that it is unrelated to any known serine hydrolases but contains a novel C-terminal domain which is conserved from bacteria to man. Furthermore, the catalytic serine is located within this domain at the centre of a helical hydrophobic segment of the polypeptide’s secondary structure. The integrity of NTE would be severely compromised by the presence of a negatively-charged organophosphate moiety at this site. Implications for possible higher-order structures and functions for NTE are discussed.

Molecular cloning, expression and site-directed mutagenesis of glutathione S-transferase from Ochrobactrum anthropi.

The gene coding for a novel glutathione S-transferase (GST) has been isolated from the bacterium Ochrobactrum anthropi. A PCR fragment of 230 bp was obtained using oligonucleotide primers deduced from N-terminal and 'internal' sequences of the purified enzyme. The gene was obtained by screening of a genomic DNA partial library from O. anthropi constructed in pBluescript with a PCR fragment probe. The gene encodes a protein (OaGST) of 201 amino acids with a calculated molecular mass of 21738 Da. The product of the gene was expressed and characterized; it showed GST activity with substrates 1-chloro-2, 4-dinitrobenzene (CDNB), p-nitrobenzyl chloride and 4-nitroquinoline 1-oxide, and glutathione-dependent peroxidase activity towards cumene hydroperoxide. The overexpressed product of the gene was also confirmed to have in vivo GST activity towards CDNB. The interaction of the recombinant GST with several antibiotics indicated that the enzyme is involved in the binding of rifamycin and tetracycline. The OaGST amino acid sequence showed the greatest identity (45%) with a GST from Pseudomonas sp. strain LB400. A serine residue in the N-terminal region is conserved in almost all known bacterial GSTs, and it appears to be the counterpart of the catalytic serine residue present in Theta-class GSTs. Substitution of the Ser-11 residue resulted in a mutant OaGST protein lacking CDNB-conjugating activity; moreover the mutant enzyme was not able to bind Sepharose-GSH affinity matrices.

Two different amino acid substitutions in the ali-esterase, E3, confer alternative types of organophosphorus insecticide resistance in the sheep blowfly, Lucilia cuprina

Two types of organophosphorus (OP) insecticide resistance are associated with reduced `ali-esterase' (E3 isozyme) activity in Lucilia cuprina. The `diazinon' resistance type shows generally greater resistance for diethyl than dimethyl OPs but no resistance to malathion. The `malathion' resistance type shows generally greater resistance for dimethyl than diethyl OPs, low level diazinon resistance, but exceptionally high malathion resistance (600 × susceptible), the last being attributed to hydrolysis of the carboxylester groups which are peculiar to malathion (malathion carboxylesterase, MCE). E3 variants from diazinon resistant strains have previously been shown to have a Gly 137 → Asp substitution that structural modelling predicts is only about 4.6 Å from the γ oxygen of the catalytic serine residue. Here we show that E3 variants from malathion resistant strains have a Trp 251 → Leu substitution predicted to be about 4.3 Å from that serine. We have expressed alleles of the gene encoding both resistance variants of E3 and an OP susceptible variant in a baculovirus system and compared the kinetics of their products. We find that both resistance substitutions reduce ali-esterase activity and enhance OP hydrolase activity. Furthermore the Gly 137 → Asp substitution enhances OP hydrolase activity for a diethyl OP substrate (chlorfenvinphos) more than does the Trp 251 → Leu substitution, which is consistent with the OP cross-resistance patterns. Trp 251 → Leu also reduces the K m for carboxylester hydrolysis of malathion about 10-fold to 21 μM, which is consistent with increased MCE activity in malathion resistant strains. We then present a model in which the malathion carboxylesterase activity of the E3-Leu 251 enzyme is enhanced in vivo by its OP hydrolase activity. The latter activity enables it to reactivate after phosphorylation by malaoxon, the activated form of malathion, accounting for the exceptionally high level of resistance to malathion. We conclude that the two types of resistance can be explained by kinetic changes caused by the two allelic substitutions in the E3 enzyme.

Probing the Abilities of Synthetically Useful Serine Proteases To Discriminate Remote Stereocenters. Chiral Naphthyl Aldehyde Inhibitors

The capacities of subtilisin Carlsberg (SC) and α-chymotrypsin (CT), which are representative of synthetically useful serine proteases, to discriminate between R- and S-configurations of stereocenters remote from the catalytic site have been further explored using chiral naphthyl aldehyde transition state analog inhibitors as probes. The inhibitors evaluated were (3R)- and (3S)-3-(1-naphthyl and 2-naphthyl)butanal and (4R)- and (4S)-4-(1-naphthyl and 2-naphthyl)pentanal, for which the methyl groups at the C-3 and C-4 stereocenters, respectively, are significantly removed from the aldehyde functionality that interactswith the catalytic serine residue. Each aldehyde was a competitive inhibitor for both enzymes, with CT being significantly more powerfully inhibited than SC. While only low levels of stereoselectivity were observed with SC, significant stereocenter discrimination was manifest for CT within this series of inhibitors with, encouragingly, the similar degrees of stereoselectivity (up to 3.9-fold) ...

Probing the Abilities of Synthetically Useful Serine Proteases To Discriminate between the Configurations of Remote Stereocenters Using Chiral Aldehyde Inhibitors

The abilities of the synthetically useful serine proteases, subtilisin Carlsberg (SC) and α-chymotrypsin (CT), to discriminate between R- and S-configurations of stereocenters remote from the catalytic site have been explored using chiral aldehyde transition state analog inhibitors as probes. The inhibitors evaluated were (R)- and (S)-3-phenylbutanal and (R)- and (S)-4-phenylpentanal, for which the stereocenters at C-3 and C-4 respectively are distant from the aldehyde functionality that interacts with the catalytic serine residue. The achiral parent compounds, 3-phenylpropanal and 4-phenylbutanal, respectively, were also assessed for reference purposes. Each aldehyde was found to be a competitive inhibitor for both enzymes, with CT being significantly more potently inhibited than SC. Within this series, the presence of an R-center methyl group improved binding significantly over that of the achiral parent aldehyde for both enzymes. In contrast, the effects on binding of S-methyl substituents in the same ...

The Vibrio cholerae hlyC gene encodes a protein that is related to lipases of pseudomonas species.

The nucleotide sequence of the Vibrio cholerae N16961 hlyC gene was determined. The hlyC gene encompasses 513 nucleotides that are predicted to encode a 171-amino acid protein with a calculated molecular weight of 18.2 kDa. The predicted HlyC protein contains a region that is 93.5% similar to the substrate-binding/catalytic domain of the Pseudomonas species triacylglycerol acylhydrolase (lipase). The proposed catalytic serine residue is also conserved in the HlyC protein. The contribution of the putative HlyC lipase to the physiology of V. cholerae is currently under investigation.

Cloning and sequence analysis of cDNA for a human homolog of eubacterial ATP-dependent Lon proteases

Overlapping cDNA clones containing mRNA for a putative Lon protease (LonHS) were isolated from cDNA libraries prepared from human brain poly(A) + RNA. The determined nucleotide sequence contains a 2814-bp open reading frame with two potential initiation codons (positions 62–64 and 338–340). The 5'-terminal 337-nucleotide fragment of LonHS mRNA is highly enriched with G and C nucleotides and could direct synthesis of the LonHS N-terminal domain. More likely this region promotes initiation of protein synthesis from the second AUG codon in a cap-independent manner. The amino acid sequence initiated at the second AUG codon includes 845 residues, over 30% of which are identical to those of eubacterial Lon proteases. Residues of the ‘A’ and ‘B’ motifs of NTP-binding pattern and a plausible catalytic serine residue are conserved in LonHS. Northern blot analysis revealed LonHS mRNA in lung, duodenum, liver and heart, but not in thymus cells.

Analysis of the structure of Pseudomonas glumae lipase.

The lipase produced by Pseudomonas glumae is monomeric in the crystalline state and has a serine protease-like catalytic triad; Ser87-His285-Asp263. The largest domain of the protein resembles closely a subset of the frequently observed alpha/beta-hydrolase fold and contains a well-defined calcium site. This paper describes structural analysis of this protein, focusing on (i) structural comparison with the lipase from Geotrichum candidum, (ii) the probable nature of the conformational change involved in substrate binding and (iii) structural variations amongst the family of Pseudomonas lipases. This analysis reveals similarities between P. glumae lipase and G. candidum lipase involving secondary structural elements of the hydrolase core and the loops carrying the catalytic serine and histidine residues. A possible functional equivalence has also been identified between parts of the two molecules thought to be involved in a conformational change. In addition, determination of the structure of P. glumae lipase has allowed rationalization of previously reported protein engineering experiments, which succeeded in improving the stability of the enzyme with respect to proteolysis.

Transcriptional regulation of an unusual trypsin-related protein expressed during insect metamorphosis

The full-length cDNA for a trypsin-related protein expressed during larval-pupal metamorphosis was obtained. The encoded N-terminal half of the protein possessed similarity to trypsin proteases, including the correctly positioned histidine and aspartic acid members of the catalytic triad. However, the remainder of the encoded protein bore little resemblance to trypsin, and was altogether missing the canonical sequence containing the catalytic serine residue. The product of in vitro transcription/translation of the cDNA was a 28 kDa protein. Under normal conditions of a declining juvenile hormone titer, transcription of the mRNA as a proportion of total genomic transcription steadily increased for the first 3 days of the final stadium, but this increase was delayed and suppressed by maintenance of a high juvenile hormone titer. During the normal increase in transcription on day 3 of the final stadium, a sharp decline was observed in the steady-state abundance of the transcript, measured relative to abundance of the remaining mRNAs, suggesting that the stability of the message decreases after tissue commitment for a pupal molt.

Catalytic subunits of the multicatalytic proteinase

The multicatalytic proteinase (MCP) has been found in a wide variety of eukaryotic cells [1,2], and a similar enzyme has been identified in archaebacteria [ 3 ] . MCP is a high molecular mass (700 kDa) non-lysosomal proteinase which may be involved in both ubiquitin-dependent and ubiquitin-independent pathways of intracellular protein degradation. The proteinase complex isolated from rat liver seems to be composed of approximately 20 different components with molecular masses ranging from 22 to 34 kDa and isoelectric points ranging from 5 to 8.5 [41. The subunits appear by electron microscopy to be arranged in the shape of a hollow cylinder. The proteinase complex can cleave peptide bonds on the carboxyl side of basic, hydrophobic and acidic amino acid residues [5], and the proteolytic activities have been referred to as trypsin-like, chymotrypsin-like and peptidyl glutamyl peptide hydrolase activities, respectively. Results of studies with a variety of inhibitors have shown that these activities are catalysed at distinct sites. It is of interest to identify which components of the complex are responsible for each of the different proteolytic activities. In this study a method has been devised for selectively labelling MCP components responsible for the trypsin-like activity. The proteinase complex was isolated from fresh rat livers using a series of chromatographic steps [4]. The method of labelling involves use of a site-specific reversible inhibitor of MCP (leupeptin) and a group-specific irreversible inhibitor (N-ethylmaleimide) . Leupeptin inhibits serine and cysteine proteinases by forming a hemiacetal or thiohemiacetal with the catalytic serine or cysteine residue, respectively [7]. Leupeptin selectively inhibits the trypsin-like activity of the MCP complex in a competitive fashion at concentrations which have no effect on the other proteolytic activities. This inhibition can be reversed either by dialysis or by the addition of sodium borohydride to reduce the reactive aldehyde group. However, the recovery of activity by either of these methods was found to be slow. NEM is also an inhibitor of the trypsinlike activity of MCP [El. It also inhibits the peptidyl glutamyl peptide hydrolase activity of MCP but has little effect on the chymotrypsin-like activity. NEM is an irreversible and specific modifier of thiol groups at neutral pH, and is available in a [14C] -labelled form. Leupeptin and NEM were used together in a differential labelling protocol where MCP was first protected by leupeptin and then modified by NEM. Both inhibitors were used at concentrations which were known to fully inhibit the trypsin-like activity. Leupeptin was removed by dialysis and then [14C]NEM was added to label sites which were previously protected by leupeptin. An experiment was also carried out in the absence of leupeptin using only labelled NEM to establish how many thiol groups react with NEM. After dialysis to remove excess labelled NEM, polypeptide components of the two labelled proteinase preparations were separated by two-dimensional SDS polyacrylamide gel electrophoresis. Labelled component (s) were identified by fluorography of the dried gel. The results of these experiments showed that up to 28 moles of [l4C1NEM were incorporated in to each mole of unprotected MCP, whereas only approximately two moles of [I4C]NEM were incorporated per mole of MCP following the differential labelling protocol. These results imply protection by leupeptin of two thiol groups per MCP molecule. Fluorography of the two different labelled MCP preparations showed that almost every component of the complex can be labelled by NEM, but that in experiments designed to label only those NEM-reactive thiol groups protected by leupeptin, only one component is strongly labelled. These labelling experiments suggest that the trypsin-like activity is located in only one of the many different components of the proteinase complex. Recent results from the labelling of catalytic sites possessing the chymotrypsin-like activity also suggest that only one component is involved [9]. These findings support the view that MCP is a complex composed of distinct components which have different functions. This work was supported by the Medical Research Council. A.J.R. is a Lister Institute Research Fellow.

Subtle Differences in Active Site Structure between Bovine and Human Thrombins: ESR and Fluorescence Studies

The primary structures of bovine and human alpha-thrombins are highly homologous yet their x-ray structures are not yet complete enough to distinguish differences. In order to probe and compare their dynamic conformations in solution, we examined bovine and human alpha-thrombins with a series of active site directed fluorosulfonylphenyl spin labeled inhibitors and fluorophores which probe a region within 10-15 A of the catalytic serine residue. Overall, the nitroxide moieties were more immobilized in the bovine vs human derivatives reflecting either more apolar binding regions or steric obstructions to the motion of the nitroxide in bovine thrombin. Most of the labels which distinguish indole (apolar ligand) binding in human thrombin were found to display similar interactions in bovine thrombin, although slight differences in the general topography of this region were suggested. The two active site directed fluorophores, dansyl fluoride and p-nitrophenyl anthranilate showed differences in both lambda emmax and lambda exmax of the complexes with bovine and human-alpha-thrombin, respectively, Several of the effects observed i.e., ligand binding (indole or benzamidine) and the subtle hydrophobic interactions between the nitroxide moiety and the protein active site would be difficult to assess from an x-ray structure determination alone.

Isolation and characterization of the human hepatic lipase gene.

Overlapping bacterial phage and cosmid genomic clones were isolated spanning an area of approximately 60 kilobases that contains the human hepatic lipase (HL) gene. It is composed of 9 exons spanning approximately 35 kilobases of DNA. The entire coding regions, the 5'-flanking sequences, and the exon-intron junctions were sequenced. The intron positions correspond to those of human lipoprotein lipase and canine pancreatic lipase, supporting the concept that these genes constitute a dispersed gene family of lipases and have evolved by duplication of a common ancestral gene. A region of the HL gene, which displays a significant homology with various other lipolytic enzymes and contains the putative catalytic site serine residue of HL, was encoded by exon 4. A major transcription start site of the human HL gene was located by primer extension analysis, 43 nucleotides upstream of the translation initiation codon. Two possible promoter elements were located 25 and 63 nucleotides upstream of the transcription initiation site: a "TATA" box-like sequence, TAATA, and a sequence found in the promoter region of many liver-specific genes, AGGTTAATTATTAAT. In addition, sequences homologous to glucocorticoid and cAMP-responsive elements were identified in the 5'-nontranscribed region.

Analysis of prepro-alpha-lytic protease expression in Escherichia coli reveals that the pro region is required for activity.

The alpha-lytic protease of Lysobacter enzymogenes was successfully expressed in Escherichia coli by fusing the promoter and signal sequence of the E. coli phoA gene to the proenzyme portion of the alpha-lytic protease gene. Following induction, active enzyme was found both within cells and in the extracellular medium, where it slowly accumulated to high levels. Use of a similar gene fusion to express the protease domain alone produced inactive enzyme, indicating that the large amino-terminal pro region is necessary for activity. The implications for protein folding are discussed. Furthermore, inactivation of the protease by mutation of the catalytic serine residue resulted in the production of a higher-molecular-weight form of the alpha-lytic protease, suggesting that the enzyme is self-processing in E. coli.

Human Plasma Lecithin-Cholesterol Acyltransferase: Inhibition of the Phospholipase A2-like Activity by sn-2-Difluoroketone Phosphatidylcholine Analogues

Abstract Lecithin-cholesterol acyltransferase (LCAT) is a plasma enzyme which catalyzes the transacylation of the sn-2-fatty acid of lecithin to cholesterol, forming lysolecithin and cholesteryl ester. We have recently proposed a covalent catalytic mechanism for LCAT in which lecithin cleavage proceeds via the formation of a transition state tetrahedral adduct between the oxygen atom of the catalytic serine residue and the sn-2-carbonyl carbon atom of the substrate (Jauhiainen, M., Ridgway, N.D., and Dolphin, P.J. (1987) Biochim. Biophys. Acta 918, 175-188). This proposal is evaluated here by use of nonhydrolyzable sn-2-difluoroketone phosphatidylcholine analogues, known to inhibit calcium-dependent phospholipase A2. These compounds inhibited the calcium-independent phospholipase A2 activity of LCAT in a time and concentration dependent manner. The most potent analogues had a 100-fold higher affinity for the enzyme than the substrate, lecithin, when present within lecithin/apoA-I proteoliposomes. The inhibition was dependent upon the presence of a difluoromethylene group alpha to the sn-2-carbonyl carbon of the analogues. The inhibition is attributed to the formation of a tetrahedral adduct between the catalytic serine residue of LCAT and the sn-2-carbonyl carbon atom of the analogues which is stabilized by the electronegative fluorine atoms present upon the carbon atom alpha to the carbonyl carbon. This adduct mimics that proposed by us to occur during lecithin cleavage by LCAT, and the data substantiate the existence of this transition state adduct prior to the release of lysolecithin and formation of a fatty acylserine oxyester of the enzyme.

Human plasma lecithin-cholesterol acyltransferase. The vicinal nature of cysteine 31 and cysteine 184 in the catalytic site.

Lecithin-cholesterol acyltransferase (LCAT) is a plasma enzyme which catalyzes the transacylation of the fatty acid at the sn-2 position of lecithin to cholesterol forming lysolecithin and cholesteryl ester. The substrates for and products of this reaction are present within the plasma lipoproteins upon which the enzyme acts to form the majority of cholesteryl ester in human plasma. We proposed a covalent catalytic mechanism of action for LCAT (Jauhiainen, M., and Dolphin, P. J. (1986) J. Biol. Chem. 261, 7032-7034) in which serine and histidine residues mediate lecithin cleavage and two cysteine residues cholesterol esterification. With the aid of sulfhydryl reactive trivalent organoarsenical compounds which are specific for vicinal thiols we have probed the geometry of the catalytic site. p-Aminophenylarsendichloride noncompetitively inactivates cholesterol esterification (Ki = 0.23 mM) by LCAT via alkylation of both catalytic cysteine residues. This reagent does not significantly inactivate lecithin cleavage by LCAT. Full enzyme activity is restored by treatment with 2,3-dimercapto-1-propanesulfonic acid. Treatment of LCAT with p-bromoacetylaminophenylarsenoxide blocks the subsequent incorporation of diisopropyl fluorophosphate and iodoacetamide and inactivates both cholesterol esterification and lecithin cleavage. These activities are not restored following 2,3-dimercapto-1-propanesulfonic acid treatment. However, the reduced cysteine thiols are regenerated and can catalyze cholesteryl arachidonate formation from arachidonyl-CoA. The control reagent, bromoacetylaniline, which lacks the sulfhydryl-reactive arsenical moiety, does not inactivate LCAT nor is this reagent incorporated into the LCAT protein. We conclude that the two catalytic cysteine residues of LCAT (Cys31 and Cys184) are vicinal with a calculated distance between their sulfur atoms of 3.50-3.62 A. The additional residue alkylated by the bifunctional reagent is within the catalytic site and may represent a previously identified catalytic serine or histidine residue.

Modification of Human Thrombin: Effect on Thrombomodulin Binding

Thrombomodulin, an endothelial cell protein, binds thrombin with high affinity and alters thrombin from a procoagulant to an anticoagulant molecule. In this study, chemical and/or proteolytic modification of thrombin was carried out to identify the essential components required for its interaction with thrombomodulin. Modification of thrombin at the catalytic site serine and histidine residues, with Diisopropylfluorophosphate and Tosyl-L-lysine chloromethyl ketone, resulted in loss of clotting and amidolytic activity. Both Diisopropyl phosphoryl-thrombin and Tosyl-L-chloromethyl ketone-thrombin inhibited native-thrombin: thrombomodulin catalyzed protein C activation with Ki values of 5 nM and 6 nM respectively indicating no loss of affinity for thrombomodulin. Oxidation of tryptophan residues with N-bromosuccinimide or iodination of tyrosine residues of thrombin led to reduced clotting and amidolytic activity as well as a reduced ability to interact with thrombomodulin. Modification of arginine residues with Phenylglyoxal and 2,3,Butanedione led to loss of thrombomodulin binding affinity. Limited proteolysis of thrombin by trypsin yielded the derivative beta-thrombin which had also lost its ability to interact with thrombomodulin. Deglycosylation of thrombin did not alter its binding affinity for thrombomodulin. These results indicate that one or more tryptophan, arginine and tyrosine residues are essential for the recognition of thrombin by thrombomodulin whilst the carbohydrate side chain and the active site residues of the thrombin molecule are not involved in thrombomodulin binding.

Phosphorylation state of HMG CoA reductase affects its catalytic activity and degradation

The expressed catalytic activity of liver microsomal HMG CoA reductase, the limiting enzyme in cholesterol synthesis, is reversibly diminished by phosphorylation in vitro. In intact hepatocytes the expressed activity of HMG CoA reductase is enhanced by incubation of cells with insulin, and diminished by treatment with glucagon or with mevalonate. In the latter situations the level of total reductase activity falls following initial inactivation (phosphorylation) of the enzyme. This observation suggested that the phosphorylated form of HMG CoA reductase is more sensitive to proteolysis. HMG CoA reductase is a 97,000 dalton (97 K) integral protein of the endoplasmic reticulum with a cytosolic domain that includes the catalytic site and serine residues that may be reversibly phosphorylated. In vitro the Ca 2+-activated proteolytic enzyme, calpain, generates two catalytically-active fragments: a membrane bound 62 K and a soluble 53 K form of the enzyme which are quantified by specific immunoblot procedures. Cleavage of the native 97 K HMG CoA reductase is enhanced by pretreatment (inactivation) of microsomes with ATP (Mg 2+) and liver reductase kinase compared to microsomes pretreated with protein phosphatase. This is reflected in a loss of 97 K reductase and an increase in the soluble 53 K form of the enzyme. Degradation of HMG CoA reductase in hepatocytes is partially blocked by lysosomotropic agents and insulin. A steady state model for the turnover of proteins subject to reversible phosphorylation has been developed which recognizes fractional degradative rate constants for the phosphorylated and dephosphorylated species.

Inhibition of the functional interaction between fatty acid synthetase and thioesterase II by modification of a single cysteine thiol on the thioesterase

Medium-chain S-acyl fatty acid synthetase thioester hydrolase (thioesterase II), a discrete, monomeric, serine active-site enzyme, modifies the product specificity of the de novo lipogenic pathway by hydrolyzing the thioester bond linking the growing acyl chain to the 4′-phosphopantetheine of the fatty acid synthetase. The mechanism of interaction of thioesterase II and fatty acid synthetase has been studied by probing the thioesterase with sulfhydryl-modifying reagents. Modification of a single cysteine thiol with 5,5′-dithiobisnitrobenzoate destroyed the ability of thioesterase II to catalyze hydrolysis of s-acyl fatty acid synthetase thioesters but had no effect on the ability of the enzyme to hydrolyze the model substrate, decanoyl- s-pantetheine. The inhibition was readily reversed on removal of the thionitrobenzoate moiety from the thioesterase with dithiothreitol. The results of kinetic experiments indicated that loss of the capacity of the thioesterase to hydrolyze the natural substrate could be attributed to an inability of the 5,5′-dithiobisnitrobenzoate-modified enzyme to bind to the fatty acid synthetase. Modification of the same cysteine thiol with methyl methanethiolsulfonate did not affect the ability of thioesterase II to hydrolyze either the natural or model substrates. The results are interpreted to indicate that cysteine thiol, remote from the catalytic activesite serine residue, is present on a binding domain of the thioesterase which interfaces with the fatty acid synthetase. Modification of this thiol with the large thionitrobenzoate moiety, but not with the small CH 3S- moiety, inhibits the functional interaction either by steric hindrance or by perturbation of the polypeptide configuration at the binding domain.