Primary structure of a chymotryptic peptide containing the "active serine" of the thioesterase domain of fatty acid synthase

“Active serine” of the thioesterase domain of fatty acid synthase from the goose uropygial gland was selectively labeled with [1,3-³H]diisopropylfluorophosphate and the chymotryptic peptide containing this active serine was purified to homogeneity by a combination of gel filtration, cation exchange chromatography and high performance liquid chromatography. The primary structure of this active site peptide, Ser-Phe-Gly-Ala-Cys-Val-Ala-Phe, is remarkably homologous to the “active serine” containing peptide of human plasmin.     

Site specificity of histone H4 methylation by wheat germ protein-arginine N-methyltransferase

CNBr treatment of calf thymus [methyl-14C]histone H4, methylated in vitro with S-adenosyl-L-[methyl-14C]methionine by a highly histone-specific wheat germ protein methylase I (S-adenosyl-L-methionine:protein-L-arginine N-methyltransferase, EC 2.1.1.23), produced two peptide fragments corresponding to residues 1-83 and 84-102, with the former being radioactive. Two-dimensional peptide mapping of the chymotryptic and tryptic digest of [methyl-14C]histone H4 and analysis of the chymotryptic digest on HPLC have shown that only a single peptide is radiolabeled. In order to define the exact site of methylation (arginine residue), the radioactive peptide from the chymotryptic digest of [methyl-14C]histone H4 was further purified on HPLC by linear and then isocratic elution. The purified chymotryptic peptide was then digested with trypsin and purified on HPLC, and its amino acid composition was determined on HPLC. These results indicate that the peptide corresponding to residues 24-35 of histone H4 is radiolabeled. Since this peptide contains a single arginine residue at position 35, we have concluded that the enzyme is specific not only to the protein substrate but also to the methylation site.     

Site-specific N-glycosylation analysis of human plasma ceruloplasmin using liquid chromatography with electrospray ionization tandem mass spectrometry

Ceruloplasmin has ferroxidase activity and plays an essential role in iron metabolism. In this study, a site-specific glycosylation analysis of human ceruloplasmin (CP) was carried out using reversed-phase high-performance liquid chromatography with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). A tryptic digest of carboxymethylated CP was subjected to LC-ESI-MS/MS. Product ion spectra acquired data-dependently were used for both distinction of the glycopeptides from the peptides using the carbohydrate B-ions, such as m/z 204 (HexNAc) and m/z 366 (HexHexNAc), and identification of the peptide moiety of the glycopeptide based on the presence of the b- and y-series ions derived from the peptide. Oligosaccharide composition was deduced from the molecular weight calculated from the observed mass of the glycopeptide and theoretical mass of the peptide. Of the seven potential N-glycosylation sites, four (Asn119, Asn339, Asn378, and Asn743) were occupied by a sialylated biantennary or triantennary oligosaccharide with fucose residues (0, 1, or 2). A small amount of sialylated tetraantennary oligosaccharide was detected. Exoglycosidase digestion suggested that fucose residues were linked to reducing end GlcNAc in biantennary oligosaccharides and to reducing end and/or alpha1-3 to outer arms GlcNAc in triantennary oligosaccharides and that roughly one of the antennas in triantennary oligosaccharides was alpha2-3 sialylated and occasionally alpha1-3 fucosylated at GlcNAc.     

A collaborative endeavor to design cholinesterase-based catalytic scavengers against toxic organophosphorus esters

Wild-type human butyrylcholinesterase (BuChE) has proven to be an efficient bioscavenger for protection against nerve agent toxicity. Human acetylcholinesterase (AChE) has a similar potential. A limitation to their usefulness is that both cholinesterases (ChEs) react stoichiometrically with organophosphosphorus (OP) esters. Because OPs can be regarded as pseudo-substrates for which the dephosphylation rate constant is almost zero, several strategies have been attempted to promote the dephosphylation reaction. Oxime-mediated reactivation of phosphylated ChEs generates a turnover, but it is too slow to make pseudo-catalytic scavengers of pharmacological interest. Alternatively, it was hypothesized that ChEs could be converted into OP hydrolases by using rational site-directed mutagenesis based upon the crystal structure of ChEs. The idea was to introduce a nucleophile into the oxyanion hole, at an appropriate position to promote hydrolysis of the phospho-serine bond via a base catalysis mechanism. Such mutants, if they showed the desired catalytic and pharmacokinetic properties, could be used as catalytic scavengers. The first mutant of human BuChE that was capable of hydrolyzing OPs was G117H. It had a slow rate. Crystallographic study of the G117H mutant showed that hydrolysis likely occurs by activation of a water molecule rather than direct nucleophilic attack by H117. Numerous BuChE mutants were made later, but none of them was better than the G117H mutant at hydrolyzing OPs, with the exception of soman. Soman aged too rapidly to be hydrolyzed by G117H. Hydrolysis was however accomplished with the double mutant G117H/E197Q, which did not age after phosphonylation with soman. Multiple mutations in the active center of human and Bungarus AChE led to enzymes displaying low catalytic activity towards OPs and unwanted kinetic complexities. A new generation of human AChE mutants has been designed with the assistance of molecular modelling and computational methods. According to the putative water-activation mechanism of G117H BChE, a new histidine/aspartate dyad was introduced into the active center of human AChE at the optimum location for hydrolysis of the OP adduct. Additional mutations were made for optimizing activity of the new dyad. It is anticipated that these new mutants will have OP hydrolase activity.     

Isolation and characterization of a 60-kilodalton glycoprotein esterase from liver microsomal membranes

A glycoprotein having a subunit weight of approximately 60,000 was isolated from rabbit liver microsomes. It is a predominant component of the hepatic microsomal membrane and reacts rapidly with diisopropylphosphorofluoridate (DFP), resulting in the loss of enzymatic activity toward artificial substrates such as acyl esters of o-nitrophenols. Automated Edman degradation of this protein together with sequence analysis of peptides provided the NH2-terminal sequence of some 70 residues as follows: His-Pro-Ser- Ala-Pro-Pro-Val-Val-Asp-Thr-Val-Lys-Gly-Lys-Val- Leu-Gly-Lys-Phe-Val-Ser-Leu-Glu-Gly-Phe-Ala-Gln- Pro-Val-Ala-Val-Phe-Leu-Gly-Val-Pro-Phe-Ala-Lys- Pro-Pro-Leu-Gly-Ser-Leu-Arg-Phe-Ala-Pro-Pro-Gln- Pro-Ala-Glu-Ser-Trp-Ser-His-Val-Lys-Asn (CHO)- Thr-Thr-Ser-Tyr-Pro-Pro-Met-Cys-Ser-Ser. A carbohydrate attachment was identified at asparaginyl residue 61. The COOH-terminal peptide of the protein was isolated from two independent enzymatic digests, and its sequence was established as Arg-Glu-Thr-Glu-His-Ile-Glu-Leu. In order to isolate the DFP binding peptide, liver microsomes were labeled with [3H]DFP and the 60-kDa protein containing covalently bound DFP isolated in pure form. Following reduction and carboxymethylation, the DFP-labeled protein was fragmented with trypsin and the digest subjected to gel filtration. Digestion of the labeled peptide preparations with chymotrypsin followed by chromatography of the digest yielded two diisopropylphosphoryl (DIP) peptides. Automated Edman degradation of these peptides provided the following amino acid sequences: Gly-Glu-DIPSer- Ala-Gly-Gly-Gln-Ser-Val-Ser-Ile-Leu-Leu-Leu-Ser- Pro and Thr-Val-Ile-Gly-Asp-DIPHis-Gly-Asp-Glu-Ile-Phe. The active site serine peptide of the 60-kDa protein shows some 70% similarity to the active center region of choline esterases. While the postulated active histidyl residue in choline esterases has not been identified, it is proposed that the DFP binding histidine of the 60-kDa protein corresponds to His-438/440 of choline esterases.     

Amino acid sequence around the serine phosphorylated by casein kinase II in brain myosin heavy chain

Casein kinase II from bovine brain transfers about one mole of phosphate to a serine residue near the COOH terminus of the heavy chain of myosin isolated from bovine brain. We have purified and characterized a peptide that contains this phosphoserine. The peptide was generated by chymotryptic and thermolytic digestion and was isolated by gel filtration, Fe3+ affinity chromatography, and reverse-phase high pressure liquid chromatography. Its sequence, Leu-Glu-Leu-Ser(PO4)-Asp-Asp-Asp-Asp-Glu-Ser-Lys-Ala-Ser-(Xaa)-Ile-Asn-Glu-Thr- Gln-Pro-Pro-Gln, shows that the Ser(PO4) is in an acidic environment, as is typical for casein kinase II phosphorylation sites. The "hydrophobic repeat" typical of alpha-helical coiled-coils is absent, suggesting that the sequence is part of a non-helical "tail piece" of the heavy chain. A synthetic peptide corresponding to residues 1-9 is shown to be an effective substrate for casein kinase II.     

Efficient chymotryptic proteolysis enhanced by infrared radiation for peptide mapping

Infrared (IR) radiation was employed to enhance the efficiency of chymotryptic proteolysis for peptide mapping in this work. Protein solutions containing chymotrypsin in sealed transparent Eppendorf tubes were allowed to digest under an IR lamp at 37 degrees C. BSA and cytochrome c (Cyt- c) were digested by IR-assisted chymotryptic proteolysis to demonstrate the feasibility and performance of the novel digestion approach and the digestion time was significantly reduced to 5 min. The obtained digests were further identified by MALDI-TOF MS with the sequence coverages that were comparable to those obtained by using conventional in-solution digestion. The suitability of IR-assisted chymotryptic proteolysis to complex proteins was demonstrated by digesting human serum. The present proteolysis strategy is simple and efficient, offering great promise for high-throughput protein identification.     

Kinetic analysis of butyrylcholinesterase-catalyzed hydrolysis of acetanilides

The aryl-acylamidase (AAA) activity of butyrylcholinesterase (BuChE) has been known for a long time. However, the kinetic mechanism of aryl-acylamide hydrolysis by BuChE has not been investigated. Therefore, the catalytic properties of human BuChE and its peripheral site mutant (D70G) toward neutral and charged aryl-acylamides were determined. Three neutral (o-nitroacetanilide, m-nitroacetanilide, o-nitrophenyltrifluoroacetamide) and one positively charged (3-(acetamido) N,N,N-trimethylanilinium, ATMA) acetanilides were studied. Hydrolysis of ATMA by wild-type and D70G enzymes showed a long transient phase preceding the steady state. The induction phase was characterized by a hysteretic "burst". This reflects the existence of two enzyme states in slow equilibrium with different catalytic properties. Steady-state parameters for hydrolysis of the three acetanilides were compared to catalytic parameters for hydrolysis of esters giving the same acetyl intermediate. Wild-type BuChE showed substrate activation while D70G displayed a Michaelian behavior with ATMA as with positively charged esters. Owing to the low affinity of BuChE for amide substrates, the hydrolysis kinetics of neutral amides was first order. Acylation was the rate-determining step for hydrolysis of aryl-acetylamide substrates. Slow acylation of the enzyme, relative to that by esters may, in part, be due suboptimal fit of the aryl-acylamides in the active center of BuChE. The hypothesis that AAA and esterase active sites of BuChE are non-identical was tested with mutant BuChE. It was found that mutations on the catalytic serine, S198C and S198D, led to complete loss of both activities. The silent variant (FS117) had neither esterase nor AAA activity. Mutation in the peripheral site (D70G) had the same effect on esterase and AAA activities. Echothiophate inhibited both activities identically. It was concluded that the active sites for esterase and AAA activities are identical, i.e. S198. This excludes any other residue present in the gorge for being the catalytic nucleophile pole.     

Crystal structures of human carboxylesterase 1 in covalent complexes with the chemical warfare agents soman and tabun

The organophosphorus nerve agents sarin, soman, tabun, and VX exert their toxic effects by inhibiting the action of human acetylcholinesterase, a member of the serine hydrolase superfamily of enzymes. The current treatments for nerve agent exposure must be administered quickly to be effective, and they often do not eliminate long-term toxic side effects associated with organophosphate poisoning. Thus, there is significant need for effective prophylactic methods to protect at-risk personnel from nerve agent exposure, and protein-based approaches have emerged as promising candidates. We present the 2.7 A resolution crystal structures of the serine hydrolase human carboxylesterase 1 (hCE1), a broad-spectrum drug metabolism enzyme, in covalent acyl-enzyme intermediate complexes with the chemical weapons soman and tabun. The structures reveal that hCE1 binds stereoselectively to these nerve agents; for example, hCE1 appears to react preferentially with the 10(4)-fold more lethal PS stereoisomer of soman relative to the PR form. In addition, structural features of the hCE1 active site indicate that the enzyme may be resistant to dead-end organophosphate aging reactions that permanently inactivate other serine hydrolases. Taken together, these data provide important structural details toward the goal of engineering hCE1 into an organophosphate hydrolase and protein-based therapeutic for nerve agent exposure.     

Analysis of thyroglobulin iodination by tandem mass spectrometry using immonium ions of monoiodo- and diiodo-tyrosine

Peptides containing a monoiodo- or diiodo-tyrosine residue (monoiodo-Y, diiodo-Y) were found to generate abundant immonium ions following collision-induced dissociation at m/z 261.97 and 387.87 Da, respectively. These residue-specific marker ions are between about 140 mDa (monoiodo-Y) and 300 mDa (diiodo-Y) mass deficient relative to any other peptide fragment ions of unmodified peptides, qualifying them as highly specific marker ions for tyrosine iodination when analyzed by high resolution tandem mass spectrometry (MS/MS). Two new iodination sites (Y-364 and Y-2165) were pinpointed in bovine thyroglobulin by MS/MS using these iodotyrosine-specific marker ions and combined tryptic/chymotryptic digestion.     

Characterization of wheat gliadin proteins by combined two-dimensional gel electrophoresis and tandem mass spectrometry

A proteomics-based approach was used for characterizing wheat gliadins from an Italian common wheat (Triticum aestivum) cultivar. A two-dimensional gel electrophoresis (2-DE) map of roughly 40 spots was obtained by submitting the 70% alcohol-soluble crude protein extract to isoelectric focusing on immobilized pH gradient strips across two pH gradient ranges, i.e., 3-10 or pH 6-11, and to sodium dodecyl sulfate-polyacrylamide electrophoresis in the second dimension. The chymotryptic digest of each spot was characterized by matrix-assisted laser desorption/ionization-time of flight mass spectrometry and nano electrospray ionization-tandem mass spectrometry (MS/MS) analysis, providing a "peptide map" for each digest. The measured masses were subsequently sought in databases for sequences. For accurate identification of the parent protein, it was necessary to determine de novo sequences by MS/MS experiments on the peptides. By partial mass fingerprinting, we identified protein molecules such as alpha/beta-, gamma-, omega-gliadin, and high molecular weight-glutenin. The single spots along the 2-DE map were discriminated on the basis of their amino acid sequence traits. alpha-Gliadin, the most represented wheat protein in databases, was highly conserved as the relative N-terminal sequence of the components from the 2-DE map contained only a few silent amino acid substitutions. The other closely related gliadins were identified by sequencing internal peptide chains. The results gave insight into the complex nature of gliadin heterogeneity. This approach has provided us with sound reference data for differentiating gliadins amongst wheat varieties.     

Characterization of the catalase-peroxidase KatG from Burkholderia pseudomallei by mass spectrometry

The electron density maps of the catalase-peroxidase from Burkholderia pseudomallei (BpKatG) presented two unusual covalent modifications. A covalent structure linked the active site Trp111 with Tyr238 and Tyr238 with Met264, and the heme was modified, likely by a perhydroxy group added to the vinyl group on ring I. Mass spectrometry analysis of tryptic digests of BpKatG revealed a cluster of ions at m/z 6585, consistent with the fusion of three peptides through Trp111, Tyr238, and Met264, and a cluster at m/z approximately 4525, consistent with the fusion of two peptides linked through Trp111 and Tyr238. MS/MS analysis of the major ions at m/z 4524 and 4540 confirmed the expected sequence and suggested that the multiple ions in the cluster were the result of multiple oxidation events and transfer of CH3-S to the tyrosine. Neither cluster of ions at m/z 4525 or 6585 was present in the spectrum of a tryptic digest of the W111F variant of BpKatG. The spectrum of the tryptic digest of native BpKatG also contained a major ion for a peptide in which Met264 had been converted to homoserine, consistent with the covalent bond between Tyr238 and Met264 being susceptible to hydrolysis, including the loss of the CH3-S from the methionine. Analysis of the tryptic digests of hydroperoxidase I (KatG) from Escherichia coli provided direct evidence for the covalent linkage between Trp105 and Tyr226 and indirect evidence for a covalent linkage between Tyr226 and Met252. Tryptic peptide analysis and N-terminal sequencing revealed that the N-terminal residue of BpKatG is Ser22.     

Physical and chemical characterization of a horse serum carboxylesterase

The serine carboxylesterase from horse serum was characterized by amino acid composition, peptide mapping, molecular and subunit weights, and sequencing of the amino acids around the essential serine residue at the active site. A protocol was developed for using reversed-phase high-performance liquid chromatography as the final step to obtain homogeneous preparations of horse serum carboxylesterase. Amounts sufficient for determining the amino acid composition and for peptide maps were obtained from a partially purified starting material which contained approximately 55% carboxylesterase. The amino acid composition, like the subunit weight (70,800 +/- 1400), was similar to the corresponding values reported for other serine carboxylesterases. However, the amino acid sequence of the tryptic digest fragment containing the essential nucleophilic seryl residue differed significantly from the corresponding sequences of other mammalian serine carboxylesterases.     

Simultaneous glycosylation analysis of human serum glycoproteins by high-performance liquid chromatography/tandem mass spectrometry

Changes in the glycosylation of some serum proteins are associated with certain diseases. In this study, we performed simultaneous site-specific glycosylation analysis of abundant serum glycoproteins by LC/Qq-TOF MS of human serum tryptic digest, the albumin of which was depleted. The glycopeptide peaks on the chromatogram were basically assigned by database searching with modified peak-list text files of MS/MS spectra and then based on mass differences of glycan units from characterized glycopeptides. Glycopeptide of IgG, haptoglobin and ceruloplasmin were confirmed by means of a comparison of their retention times and m/z values with those obtained by LC/MS of commercially available glycoproteins. Mass spectrometric carbohydrate heterogeneity in the assigned glycopeptides was analyzed by an additional LC/MS. We successfully demonstrated site-specific glycosylation of 23 sites in abundant serum glycoproteins.     

Bacillus cereus 569/H penicillinase serine-44 acylation by diazotized 6-aminopenicillanic acid

Penicillinase from Bacillus cereus 569/H was purified to homogeneity. Its active site was probed by use of an affinity label generated in situ by the diazotization of 6-aminopenicillanic acid, a catalytically poor substrate for this enzyme. The loss of activity arising during the inactivation is dependent upon pH and the penicillin:sodium nitrite ratio used. Optimal inactivation was obtained at pH 4.7 and reactivation could be prevented if subsequent purification and manipulations were performed at low pH. Inactivation by diazotized 6-aminopenicillanic acid was characterized further by tryptic and chymotryptic digestion of the inactivated enzyme and peptide mapping of the resulting digests. Amino acid analysis of the chymotryptic labeled peptide yielded a composition which corresponds to residues 41-46 (Ala-Phe-Ala-Ser-Thr-Tyr) in the published partial sequence of the enzyme (Thatcher, D. (1975) Biochem. J. 147, 313-326). Further digestion of this chymotryptic peptide with carboxypeptidase A reveals that serine-44 is modified in this affinity labeling procedure. Mass spectral analysis of the modified serine residue and alkali-released label, and comparison with spectra of model compounds indicates that the inactivation occurs with rearrangement of the beta-lactamthiazolidine structure to a dihydrothiazine.     

Identification of the active site of Citrobacter freundii beta-lactamase using dansyl-penicillin

The active site sequence of a beta-lactamase encoded by chromosomal gene(s) in Citrobacter freundii GN346 was determined using dansyl-penicillin as a fluorescent probe. The tryptic digest of the labelled enzyme gave a fluorescent peptide containing 22 amino acids. The sequence of this peptide was identical to the consensus sequence of class C beta-lactamases, Gly-Ser-X-Ser-Lys. The residue labelled was the serine adjacent to the glycine. The active site sequence corresponded to positions 46-67 of the entire sequence of the Citrobacter freundii beta-lactamase determined on the basis of the DNA sequence of the structural gene [(1986) Eur. J. Biochem. 156, 441-445]. The labelled serine corresponded to Ser-64.     

Isolation of a galactose-free 20-kDa fragment exhibiting butyrylcholine esterase and aryl acylamidase activity from human serum butyrylcholine esterase by limited alpha-chymotrypsin digestion

Purified human serum butyrylcholine esterase (approximately 90-kDa subunit), which also exhibits aryl acylamidase activity, was subjected to limited alpha-chymotrypsin digestion. Three major protein fragments of approximately 50 kDa, approximately 21 kDa and approximately 20 kDa were found to be produced, as observed by SDS-gel electrophoresis of the chymotryptic digest. The purified butyrylcholine esterase could fully bind to a Ricinus-communis-agglutinin-Sepharose column but after chymotryptic digestion about 15-20% of the enzyme activity remained unbound and was recovered in the run-through fractions. Sephadex G-75 chromatography of the chymotryptic digest showed an enzymatically active fragment eluted at an approximate molecular mass of 20 kDa, apart from the undigested butyrylcholine esterase eluted at the void volume. The butyrylcholine esterase fragment that did not bind to Ricinus communis agglutinin also was eluted at an approximate molecular mass of 20 kDa from a Sephadex G-75 column. This enzymatically active low-molecular-mass fragment from Sephadex G-75 chromatography showed a single protein band of approximately 20 kDa on SDS-gel electrophoresis. Neutral sugar analysis of the approximately 20 kDa fragment showed the presence of mannose only, whereas the undigested butyrylcholine esterase showed the presence of both mannose and galactose. Amino-terminal-sequence analysis of the approximately 20 kDa fragment showed the sequence Arg-Val-Gly-Ala-Leu, which agrees with amino acid residues 147-151 reported for human serum butyrylcholine esterase [Lockridge et al. (1987) J. Biol. Chem. 262, 549-557]. Both cholinesterase and aryl acylamidase activities were co-eluted in all chromatographic procedures. The results suggested that limited alpha-chymotrypsin digestion of human serum butyrylcholine esterase resulted in the formation of a approximately 20-kDa enzymatically active fragment with Arg147 as its N-terminal residue and which was devoid of galactose.     

Autocatalytic cleavage of myelin basic protein: an alternative to molecular mimicry

Although multiple sclerosis (MS) is thought to be an autoimmune disease, the mechanisms by which immunodominant epitopes are generated and lymphocytes are activated are not known. Here, myelin basic protein-component 1 (MBP-C1) from MS tissue was shown to undergo autocatalytic cleavage at slightly alkaline pH. Importantly, one of the major peptides released contained the immunodominant epitope 84-89. Interestingly, MBP isolated from MS patients showed a faster time course of cleavage and a more robust release of epitope 84-89 than MBP isolated from normal individuals. The cleavage reaction was not inhibited by protease inhibitors, except for phenylmethanesulfonyl fluoride (PMSF), a serine protease inhibitor. Since PMSF inhibition suggested a role for a serine residue in the cleavage, we labeled myelin basic protein with diisopropyl fluorophosphate (DFP), known to bind active site serine residues. Mass spectrometry was used to identify the labeled peptide, which consisted of residues 140-152. Since this peptide contained a single serine residue, we concluded it to be the active serine. The importance of this cleavage mechanism is that it provides for a ready source of the immunodominant peptide for sensitization of T-cells. It is not necessary to invoke other mechanisms such as molecular mimicry.     

Affinity alkylation of 3-oxo-delta 5-steroid isomerase by steroidal 3 beta-oxiranes: identification of the modified amino acid by reduction with hydroxyborohydride

The steroidal 3 beta-oxirane (3S)-spiro[5 alpha-androstane-3,2'-oxiran]-17 beta-ol (1 beta) is an active site directed irreversible inhibitor of the 3-oxo-delta 5-steroid isomerase from Pseudomonas testosteroni. Two steroid-bound peptides (TPS1 and TPS2) were isolated by high-performance liquid chromatography (HPLC) from the trypsin digest of enzyme inactivated with 1 beta. The modified tryptic peptides (residues 14-45 of the enzyme) were further digested with chymotrypsin, each giving rise to a single steroid-containing product (CPS1 and CPS2, respectively) derived from residues 31 to 45 of the enzyme. The modified chymotryptic peptides were isolated by HPLC, and the peptide-steroid ester linkage was reduced with sodium hydroxyborohydride. Amino acid analysis of the reduced peptides gave ca. 0.5 residue of homoserine and one less residue of aspartic acid than the corresponding unreduced peptides. Sequence analysis of both reduced chymotryptic peptides revealed that homoserine was located at position 8 in the peptide sequence, corresponding to residue 38 of the enzyme. The finding that the steroidal 3 beta-oxirane, like the 17 beta-oxiranes, inactivates the isomerase via esterification of aspartic acid-38 is strong evidence that this enzyme binds steroids in at least two orientations.     

Amino acid sequence of the active site of Acanthamoeba myosin II

We have used the substrate [5,6-3H]UTP for direct photoaffinity labeling of the active site of the heavy chain of myosin II from Acanthamoeba castellanii. The only labeled peptide in a total tryptic digest had the sequence of Thr-Glu-Asn-Thr-Me2Lys-Lys (where Me2Lys represents dimethyllysine) with the substrate covalently bound to the Glu residue. This sequence differs at only one position from the sequence of residues 184-189 of nematode myosin heavy chain (Me2Lys----Lys), a post-translational modification, and at two additional positions from residues 185-190 of rabbit skeletal muscle myosin (Glu----Val and Lys----Arg). The partial sequence of a larger labeled peptide derived from total chymotryptic digestion was compatible with and extended this sequence. A 20-residue sequence that contains the active site, tryptic hexapeptide is otherwise identical in Acanthamoeba and rabbit skeletal muscle myosins and has only one more difference in nematode myosin. Because UTP is a substrate for myosin II and a "zero-length" probe, we believe that it identifies amino acid residues that are very close to the substrate during the catalytic cycle.