Phenylglyoxal reveals phosphorylation-dependent difference in the conformation of Acanthamoeba myosin II active site

Acanthamoeba myosin II is regulated in an unique way by phosphorylation of three serine residues located within nonhelical tailpiece of the rod domain. Phosphorylation inhibits functions associated with the NH2-terminal motor domain, i.e., actin-activated activity and ability to move actin filaments. Number of data indicate functional communication between these distant domains. In this work, effect of modification of arginine residues with phenylglyoxal on the Ca2+-ATPase activity and susceptibility to endoproteinase ArgC cleavage of monomeric phospho- and dephosphomyosin II has been investigated. Upon the phenylglyoxal treatment the activity of dephosphomyosin II was decreasing faster that the activity of phosphomyosin. The modification also affected the proteolytic fragmentation of phospho- and dephosphomyosin II: the cleavage of heavy chain was further inhibited for phosphomyosin and enhanced for dephosphomyosin with a concomitant exposure of an additional cleavage site within the head domain. No difference in the quantity of modified arginines was observed. These results indicate a difference between the conformation of active sites of phospho- and dephosphomyosin II.     

Amino acid sequence of Acanthamoeba actin

By amino acid sequence studies, only one form of cytoplasmic actin was detected in Acanthamoeba castellanii. Its amino acid sequence is very similar to the sequences of Dictyostelium and Physarum actins, from which Acanthamoeba actin differs in only nine and seven residues, respectively, including the deletion of the first residue. Acanthamoeba actin is unique in containing a blocked NH2-terminal neutral amino acid (glycine), while all other actins sequenced thus far have a blocked acidic amino acid (aspartic or glutamic) at the NH2 terminus. Acanthamoeba actin is also unique in that it contains an N epsilon-trimethyllysine residue at position 326. Like other actins, Acanthamoeba actin contains an NT-methylhistidine residue at position 73. The protein sequence is in complete agreement with the sequence derived from the nucleotide sequence of an expressed actin gene.     

Amino acid sequence of a segment of the Acanthamoeba myosin II heavy chain containing all three regulatory phosphorylation sites

The actin-activated Mg2+-ATPase activity of myosin II from the soil amoeba Acanthamoeba castellanii is regulated by phosphorylation of 3 serine residues on the myosin II heavy chain. Partial chymotryptic digestion of 32P-labeled myosin II cleaves from the tail end of the myosin II heavy chain a small peptide which contains all three phosphorylation sites. During purification the phosphorylated peptide is resolved into several different species as a result of heterogeneity both in phosphate content and in size (probably due to chymotryptic cleavage at the carboxyl terminus). However, all forms of the peptide have an identical amino terminus. The sequence of the first 58 residues of the peptide is: N-S-A-L-E-S-D-K-Q-I10-L-E-D-E-I-G-D-L-H- E20-K-N-K-Q-L-Q-A-K-I-A30-Q-L-Q-D-E-I-D-G-T- P40-S-S-R-G-G-S-T-R-G-A50-S-A-R-G-A-S-V-R. The phosphorylated serines are at positions 46, 51, and 56. The first 36 residues of the sequence display a repeating 3-4-3-4 pattern of hydrophobic residues suggesting that this section of the peptide forms an alpha-helical coiled-coil structure. A -Gly-Thr-Pro sequence at residues 38-40 disrupts the alpha-helix and, at the same point, the repeating pattern of non-polar residues is lost. It is likely that the residues extending from Gly-38 to the end of the myosin II tail, which include the 3 phosphorylatable serines, form a randomly coiled or small globular structure. This is the first report of the sequence around the regulatory phosphorylation sites on any myosin heavy chain.     

Amino acid sequence of the active site of human pseudocholinesterase

The usualE ₁ ^u and atypicalE ₁ ^a human pseudocholinesterases (acylocholine acylhydrolase, EC 3.1.1.8) were purified to homogeneity. The active-site serine residue was conjugated with diisopropyl fluorophosphate and digested with trypsin. The tryptic peptide containing the active site was isolated by gel filtration followed by two-dimensional paper chromatography and electrophoresis. The amino acid sequence of the active site peptide obtained from the usualE ₁ ^u enzyme was found to be Gly-Glu-Ser-Ala-Gly-Ala-Ser-Ala-Val-Ser-Leu. A remarkable structural homology exists between the human and the horse enzymes in their active sites. From the difference in electrophoretic mobility of the active-site peptides obtained from the usual and atypical enzymes, the probable structure of the atypical human enzyme was deduced as Gly-His-Ser-Ala-Gly-Ala-Ser-Ala-Val-Ser-Leu.     

Amino acid sequence of the phosphorylation site of bovine cardiac myosin light chain

Amino acid sequences of peptides containing the phosphorylation site of bovine cardiac myosin light chain (L2) were determined. The site was localized to a serine residue in the tentative amino terminus of the light chain and is homologous to phosphorylation sites in other myosin light chains. Phosphorylation of bovine cardiac light chain by chicken gizzard myosin light chain kinase was Ca2+-calmodulin dependent. Kinetic data gave a Km of 107; microM and a Vmax of 23.6 mumol min-1 mg-1. In contrast to what has been observed with smooth muscle light chains, neither the phosphorylation site fragment of the cardiac light chain nor a synthetic tetradecapeptide containing the phosphorylation site were effectively phosphorylated by the chicken gizzard kinase. Phosphorylation of cardiac myosin light chains by chicken gizzard myosin light chain kinase, therefore, requires other regions of the light chain in addition to a phosphate acceptor site.     

Amino acid sequence at the active site of beta-glucosidase A from bitter almonds.

beta-Glucosidase A from bitter almonds was inhibited by the substrate analogue 6-bromo-3,4,5-trihydroxycyclo[2-3H]hex-1-ene oxide. Incorporation of 2 mol inhibitor/mol of dimeric enzyme resulted in total loss of activity. From tryptic digests of the labeled enzyme two radioactive peptides were isolated and their sequence determined (binding site of inhibitor underlined): peptide I, containing approx. 60% of the label: Ile-Thr-Glx-Glx-Gly-Val--Phe-Gly-Asp-Ser-Glx-(Ala, Asx2, Pro)-Lys and peptide II with approx. 30% of the label: Gly-Thr-Glx-Asp. The specifity of the reaction of beta-glucosidases (beta-D-glucoside glucohydrolase, EC 3.2.1.21) with substrate-related epoxides indicates that the aspartic acid labeled in peptide I participates in the catalytic process of beta-glucoside hydrolysis. The labeling of a second site is interpreted in terms of two, mutually exclusive, binding modes of the inhibitor.     

Amino acid sequence of an active fragment of rabbit skeletal muscle myosin light chain kinase

The amino acid sequence of a 368-residue segment at the carboxyl-terminus of rabbit skeletal muscle myosin light chain kinase (MLCK) has been determined. The sequence was derived primarily from analysis of two complementary sets of fragments obtained by cleavage at methionyl and arginyl bonds in S-carboxymethylated MLCK. The segment included a 360-residue fragment produced by limited tryptic digestion of MLCK. This fragment was both catalytically active and dependent on Ca2+-calmodulin. Unique structural features of MLCK have been identified, and a likely calmodulin interaction site is suggested. Sequence comparisons of MLCK to other protein kinases indicate close structural relationships in spite of marked differences in physicochemical properties, enzymatic characteristics, and regulatory response among these enzymes.     

Amino acid sequence of the active site peptide of bovine intestinal 5'-nucleotide phosphodiesterase and identification of the active site residue as threonine

We report here the identification of the amino acid residue which forms the covalent intermediate in the catalytic mechanism of bovine intestinal 5'-nucleotide phosphodiesterase and the sequence of the neighboring amino acids. The active site of 5'-nucleotide phosphodiesterase was labeled using thymidine 5'-[alpha-32P]triphosphate as substrate. A single labeled cyanogen bromide peptide was isolated using reversed-phase high performance liquid chromatography. After subdigestion with endoproteinase Lys-C and chymotrypsin, the entire amino acid sequence of the 60-residue active site peptide was obtained using automated Edman degradation. All of the radioactivity of the active site peptide was localized to a hexapeptide with sequence Thr-Phe-Pro-Asn-His-Tyr. Phosphoamino acid analysis of this peptide indicated that the labeled residue was threonine. We are not aware of any other enzymes in which threonine is phosphorylated as a covalent intermediate in the catalytic mechanism.     

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.     

Amino acid sequence around the active site cysteine residue of calcium-activated neutral protease (CANP)

We have examined hydrophobic properties of Tetrahymena CaM using the uncharged probe, n-phenyl-1-naphthylamine (NPN) fluorescence. The maximal fluorescence intensity of Tetrahymena calmodulin (CaM) is less than 1/12 of that of the bovine brain CaM. In the phosphodiesterase activation, the potency of Tetrahymena CaM, which was represented by reciprocals of the quantity of CaM required for half-maximal activation of enzyme was 22.7% respectively, of that of the bovine brain CaM. Here, Tetrahymena CaM had less hydrophobic groups exposed in the presence of Ca2+. Then Ca2+-CaM dependent enzymes require much amount of Tetrahymena CaM, comparing with the bovine brain CaM.     

Amino acid sequence of protein B23 phosphorylation site

A major phosphopeptide labeled in vivo, was identified in nucleolar protein B23 (Mr/pI = 37,000/5.1) after tryptic digestion. This peptide was purified by high performance liquid chromatography using reverse-phase (C8 and C18) columns. The phosphopeptide contains 20 amino acids including 1 phosphoserine, 7 glutamic acids, and 4 aspartic acids. The amino acid sequence is: His-Leu-Val-Ala-Val-Glu-Glu-Asp-Ala-Glu-Ser(P)-Glu-Asp-Glu-Asp- Glu-Glu-Asp-Val-Lys. This amino acid sequence is similar to that of nucleolar phosphoprotein C23 (8 consecutive amino acids were identical), and to the regulatory subunit (RII) of cAMP-dependent protein kinase (7 consecutive amino acids were identical, which is phosphorylated by casein kinase II (Carmichael, D.F., Geahlen, R.L., Allen, S.M., and Krebs, E.G. (1982) J. Biol. Chem 257, 10440-10445). The regions near these phosphorylation sites are enriched with glutamic and aspartic acids, suggesting that this acidic amino acid cluster may be essential for kinase recognition.     

Amino acid sequence of rabbit skeletal muscle myosin light chain kinase

The amino acid sequence of the amino-terminal, 235-residue segment of rabbit skeletal muscle myosin light chain kinase has been determined. Together with the carboxyl-terminal segment previously described [Takio, K., Blumenthal, D. K., Edelman, A. M., Walsh, K. A., Krebs, E. G., & Titani, K. (1985) Biochemistry 24, 6028], the present work completes the 603-residue sequence of this protein. The amino-terminal segment that has been analyzed herein corresponds to a domain reported to be of highly asymmetrical shape and as yet unknown function. Secondary structure calculations failed to provide any evidence of alpha-helix or beta-structures, but polyproline II like helical structure is possible. Sequence analysis indicates the presence of approximately equal quantities of two isoforms differing in a single amino acid replacement. Unexpected difficulties were encountered in the present sequence analysis due to the presence of acid-labile Asp-Pro bonds and to five separable variants of a blocked 21-residue amino-terminal peptide, arising from rearrangement at an Asn-Gly bond.     

Amino acid sequence of the regulatory light chain of squid mantle muscle myosin

The amino acid sequence of the regulatory light chain of mantle muscle myosin from squid (Todarodes pacificus) was determined by conventional methods. It was: xA-E-E-A-P-R-R-V-K-L-S-Q-R-Q-M-Q-E-L-K-E-A-F-T-M-I-D-Q-D-R-D-G-F-I-G-M- E-D-L-K-D-M-F-S-S-L-G-R-V-P-P-D-D-E-L-N-A-M-L-K-E-C-P-G-Q-L-N-F-T- A-F-L-T-L-F-G-E-K-V-S-G-T-D-P-E-D-A-L-R-N-A-F-S-M-F-D-E-D-G-Q-G-F-I-P- E-D-Y-L-K-D-L-L-E-N-M-G-D-N-F-S-K-E-E-I-K-N-V-W-K-D-A-P-L-K-N-K-Q-F- N-Y-N-K-M-V-D-I-K-G-K-A-E-D-E-D. The alpha-amino group of this light chain was blocked, and a typical calcium-binding structure was recognized at the sequence of residue 26 to residue 37, like those in other myosin regulatory light chains.