Synthetic peptide substrates for the erythrocyte protein carboxyl methyltransferase. Detection of a new site of methylation at isomerized L-aspartyl residues

Four hexapeptides of sequence L-Val-L-Tyr-L-Pro-(Asp)-Gly-L-Ala containing D- or L-aspartyl residues in normal or isopeptide linkages have been synthesized by the Merrifield solid-phase method as potential substrates of the erythrocyte protein carboxyl methyltransferase. This enzyme has been shown to catalyze the methylation of D-aspartyl residues in proteins in red blood cell membranes and cytosol. Using a new vapor-phase methanol diffusion assay, we have found that the normal hexapeptides containing either D- or L-aspartyl residues were not substrates for the human erythrocyte methyltransferase. On the other hand, the L-aspartyl isopeptide, in which the glycyl residue was linked in a peptide bond to the beta-carboxyl group of the aspartyl residue, was a substrate for the enzyme with a Km of 6.3 microM and was methylated with a maximal velocity equal to that observed when ovalbumin was used as a methyl acceptor. The enzyme catalyzed the transfer of up to 0.8 mol of methyl groups/mol of this peptide. Of the four synthetic peptides, only the L-isohexapeptide competitively inhibits the methylation of ovalbumin by the erythrocyte enzyme. This peptide also acts as a substrate for both of the purified protein carboxyl methyltransferases I and II which have been previously isolated from bovine brain (Aswad, D. W., and Deight, E. A. (1983) J. Neurochem. 40, 1718-1726). The L-isoaspartyl hexapeptide represents the first defined synthetic substrate for a eucaryotic protein carboxyl methyltransferase. These results demonstrate that these enzymes can not only catalyze the formation of methyl esters at the beta-carboxyl groups of D-aspartyl residues but can also form esters at the alpha-carboxyl groups of isomerized L-aspartyl residues. The implications of these findings for the metabolism of modified proteins are discussed.     

Nucleotide sequence of the yeast STE14 gene, which encodes farnesylcysteine carboxyl methyltransferase, and demonstration of its essential role in a-factor export.

Eukaryotic proteins initially synthesized with a C-terminal CAAX motif (C is Cys, A is aliphatic, and X can be one of several amino acids) undergo a series of modifications involving isoprenylation of the Cys residue, proteolysis of AAX, and alpha-carboxyl methyl esterification of the newly formed isoprenyl cysteine. We have previously demonstrated that STE14 encodes the enzyme which mediates carboxyl methylation of the Saccharomyces cerevisiae CAAX proteins a-factor, RAS1, and RAS2. Here we report the nucleotide sequence of STE14, which indicates that STE14 encodes a protein of 239 amino acids, predicted to contain multiple membrane-spanning segments. Mapping data indicate that STE14 resides on chromosome IV, tightly linked to ADE8. By analysis of ste14 null alleles, we demonstrated that MATa ste14 mutants are unable to mate but are viable and exhibit no apparent growth defects. Additional analysis of ste14 ras 1 and ste14 ras2 double mutants, which grow normally, reinforces our previous conclusion that RAS function is not significantly influenced by its methylation status. We examine a-factor biogenesis in a ste14 null mutant by metabolic labeling and immunoprecipitation and demonstrate that although proteolytic processing and membrane localization of a-factor are normal, the ste14 null mutant exhibits a profound block in a-factor export. This observation suggests that the methyl group is likely to be a critical recognition determinant for the a-factor transporter, STE6, thus providing insight into the substrate specificity of STE6 and also supporting the hypothesis that carboxyl methylation can have a dramatic impact on protein-protein interactions.     

Enzyme-substrate interactions in the hydrolysis of peptide substrates by thermitase, subtilisin BPN', and proteinase K

Peptide substrates of the general structure acetyl-Alan (n = 2-5), acetyl-Pro-Ala-Pro-Phe-Alan-NH2 (n = 0-3), and acetyl-Pro-Ala-Pro-Phe-AA-NH2 (AA = various amino acids) were synthesized and used to investigate the enzyme-substrate interactions of the microbial serine proteases thermitase, subtilisin BPN', and proteinase K on the C-terminal side of the scissile bond. The elongation of the substrate peptide chain up to the second amino acid on the C-terminal side (P'2) enhances the hydrolysis rate of thermitase and subtilisin BPN', whereas for proteinase K an additional interaction with the third amino acid (P'3) is possible. The enzyme subsite S'1 specificity of the proteases investigated is very similar. With respect to kcat/Km values small amino acid residues such as Ala and Gly are favored in this position. Bulky residues such as Phe and Leu were hydrolyzed to a lower extent. Proline in P'1 abolishes the hydrolysis of the substrates. Enzyme-substrate interactions on the C-terminal side of the scissile bond appear to affect kcat more than Km for all three enzymes.     

Protease La, the lon gene product, cleaves specific fluorogenic peptides in an ATP-dependent reaction

Protease La is an ATP-dependent protease that catalyzes the rapid degradation of abnormal proteins and certain normal polypeptides in Escherichia coli. In order to learn more about its specificity and the role of ATP, we tested whether small fluorogenic peptides might serve as substrates. In the presence of ATP and Mg2+, protease La hydrolyzes two oligopeptides that are also substrates for chymotrypsin, glutaryl-Ala-Ala-Phe-methoxynaphthylamine (MNA) and succinyl-Phe-Leu-Phe-MNA. Methylation or removal of the acidic blocking group prevented hydrolysis. Closely related peptides (glutaryl-Gly-Gly-Phe-MNA and glutaryl-Ala-Ala-Ala-MNA) are cleaved only slightly, and substrates of trypsin-like proteases are not hydrolyzed. Furthermore, several peptide chloromethyl ketone derivatives that inhibit chymotrypsin and cathepsin G (especially benzyloxycarbonyl-Gly-Leu-Phe-chloro-methyl ketone), inhibited protease La. Thus its active site prefers peptides containing large hydrophobic residues, and amino acids beyond the cleavage site influence rates of hydrolysis. Peptide hydrolysis resembles protein breakdown by protease La in many respects: 1) ADP inhibits this process rapidly, 2) DNA stimulates it, 3) heparin, diisopropyl fluorophosphate, and benzoyl-Arg-Gly-Phe-Phe-Leu-MNA inhibit hydrolysis, 4) the reaction is maximal at pH 9.0-9.5, 5) the protein purified from lon- E. coli or Salmonella typhymurium showed no activity against the peptide, and that from lonR9 inhibited peptide hydrolysis by the wild-type enzyme. With partially purified enzyme, peptide hydrolysis was completely dependent on ATP. The pure protease hydrolyzed the peptide slowly when only Mg2+, Ca2+, or Mn2+ were present, and ATP enhanced this activity 6-15-fold (Km = 3 microM). Since these peptides cannot undergo phosphorylation, adenylylation, modification of amino groups, or denaturation, these mechanisms cannot account for the stimulation by ATP. Most likely, ATP and Mg2+ affect the conformation of the enzyme, rather than that of the substrate.     

In vivo differential prenylation of retinal cyclic GMP phosphodiesterase catalytic subunits.

A number of phototransducing proteins in vertebrate photoreceptors contain a carboxyl terminal -CXXX motif (where C = cysteine and X = any amino acid), known to be a signal sequence for their post-translational prenylation and carboxyl methylation. To study the roles of these modifications in the visual excitation process, we have utilized an intravitreal injection method to radiolabel the prenylated proteins of rat retinas in vivo. We showed that two of the major prenylated polypeptides in the rod outer segments are the PDE alpha and PDE beta subunits of cyclic GMP phosphodiesterase PDE alpha and PDE beta subunits of cyclic GMP phosphodiesterase (PDE). By chromatographic analyses of the amino acid constituents generated by exhaustive proteolysis of PDE alpha and PDE beta, we further demonstrated that they are differentially prenylated by farnesylation and geranylgeranylation, respectively. While a number of proteins ending with the -CXXX sequence have already been reported to possess either a farnesyl or a geranylgeranyl group, PDE is the first enzyme shown to be modified by both types of prenyl groups. The prenyl modification of PDE most likely plays a major role in membrane attachment and in correctly positioning the PDE molecule for phototransduction.     

Structural elements affecting the recognition of L-isoaspartyl residues by the L-isoaspartyl/D-aspartyl protein methyltransferase. Implications for the repair hypothesis.

We have synthesized a series of L-isoaspartyl-containing (isoD) peptides and characterized their interaction with the human erythrocyte L-isoaspartyl/D-aspartyl protein methyltransferase (EC 2.1.1.77). Our findings indicate that this enzyme interacts with 6 residues extending from the isoD-2 to isoD+3 positions in peptide substrates. Although peptides as simple as G-isoD-G are methylated with low affinity (Km = 17.8 mM), a wide variety of L-isoaspartyl-containing sequences in larger peptides are recognized with high affinity (Km less than 20 microM), the best yet discovered being VYP-isoD-HA, with a Km of 0.29 microM. Only two sequence elements have been found that can interfere with the high affinity binding of peptides of 4 or more residues, these being a prolyl residue in the isoD+1 position and negatively charged residues in the isoD+1, isoD+2, and/or isoD+3 positions. We investigated the effect of higher order structure on binding affinity using several L-isoaspartyl-containing proteins. Although conformation did, in some cases, lower the affinity of the methyltransferase for L-isoaspartyl residues, the range of kinetic constants for the methylation of these proteins was similar to that observed with the synthetic peptides. The L-isoaspartyl/D-aspartyl methyltransferase has been proposed to function in vivo to prevent the accumulation of L-isoaspartyl residues that arise spontaneously as proteins age. To examine whether such a mechanism is feasible given the wide range of substrate Km values observed in vitro, we set up a computer simulation to model the degradation and methylation reactions in aging human erythrocytes. Our results suggest that enough methyltransferase activity exists in these cells to significantly lower the expected number of L-isoaspartyl residues, even when these residues have millimolar Km values for methylation.     

Characterization of the platelet-derived growth factor beta-receptor kinase activity by use of synthetic peptides

Synthetic peptides derived from the sequence surrounding tyrosine-857 in the human platelet-derived growth factor (PDGF) beta-receptor were used to elucidate the requirement for length and presence of negative and positively charged amino acids in substrates of the PDGF beta-receptor protein tyrosine kinase. The measured Km for the different peptides were all in the range 1-10 mM. A peptide of only five amino acids, lacking acidic amino acid residues, were found to be substrates for the receptor kinase. Ligand binding was found to stimulate the phosphorylation of peptides mainly by lowering the Km both for peptide and for ATP. Only minor changes in the Vmax occurred upon stimulation with PDGF. The reaction mechanism was found to be sequential, i.e. both the peptide and ATP have to bind to the enzyme before any product is released.     

Synthetic peptides and fluorogenic substrates related to the reactive site sequence of Kunitz-type inhibitors isolated from Bauhinia: interaction with human plasma kallikrein

We have previously described Kunitz-type serine proteinase inhibitors purified from Bauhinia seeds. Human plasma kallikrein shows different susceptibility to those inhibitors. In this communication, we describe the interaction of human plasma kallikrein with fluorogenic and non-fluorogenic peptides based on the Bauhinia inhibitors' reactive site. The hydrolysis of the substrate based on the B. variegata inhibitor reactive site sequence, Abz-VVISALPRSVFIQ-EDDnp (Km 1.42 microM, kcat 0.06 s(-1), and kcat/Km 4.23 x 10(4) M(-1) s(-1)), is more favorable than that of Abz-VMIAALPRTMFIQ-EDDnp, related to the B. ungulata sequence (Km 0.43 microM, kcat 0.00017 s(-1), and kcat/Km 3.9 x 10(2) M(-1) s(-1)). Human plasma kallikrein does not hydrolyze the substrates Abz-RPGLPVRFESPL-EDDnp and Abz-FESPLRINIIKE-EDDnp based on the B. bauhinioides inhibitor reactive site sequence, the most effective inhibitor of the enzyme. These peptides are competitive inhibitors with Ki values in the nM range. The synthetic peptide containing 19 amino acids based on the B. bauhinioides inhibitor reactive site (RPGLPVRFESPL) is poorly cleaved by kallikrein. The given substrates are highly specific for trypsin and chymotrypsin hydrolysis. Other serine proteinases such as factor Xa, factor XII, thrombin and plasmin do not hydrolyze B. bauhinioides inhibitor related substrates.     

Purification and characterization of cobyrinic acid a,c-diamide synthase from Pseudomonas denitrificans.

Cobyrinic acid a,c-diamide synthase, which catalyzes the conversion of cobyrinic acid to cobyrinic acid a,c-diamide via the intermediate formation of cobyrinic acid c-monoamide, was purified 155-fold to homogeneity from extracts of a recombinant strain of Pseudomonas denitrificans by high-performance liquid chromatography. The enzyme has an apparent molecular weight of 86,000 and consists of two identical subunits of Mr 45,000, as estimated by gel electrophoresis under denaturing conditions. Stepwise Edman degradation provided the N-terminal sequence of the first 15 amino acids. Glutamine was shown to be the preferred amino group donor (Km = 20.3 microM), but it could be replaced by ammonia (Km = 12 mM). The reaction was ATP dependent and exhibited a broad optimum pH around 7.3. Km values for (CN,aq)cobyrinic acid, (aq)2cobyrinic acid, and (CN,aq)cobyrinic acid c-monoamide were 160, greater than or equal to 250, and 71 microM, respectively. Hydrogenobyrinic acid and hydrogenobyrinic acid c-monoamide were shown to be much better substrates, with Km values of 0.41 and 0.21 microM, respectively.     

RAS2 protein of Saccharomyces cerevisiae undergoes removal of methionine at N terminus and removal of three amino acids at C terminus

RAS2 protein of Saccharomyces cerevisiae undergoes post-translational modifications involving methyl esterification and palmitic acid addition, resulting in their association with the plasma membrane. In this paper, we provide evidence that two kinds of proteolytic events accompany the biosynthesis. This is shown by separating and characterizing three intracellular forms of RAS2 protein: precursor, intermediate, and mature (fatty acid-acylated) forms. N-Terminal sequencing has revealed that all three forms start with proline, which is the second amino acid expected from the RAS2 gene sequence. Thus, the first methionine is removed very early during the biosynthesis. Isolation and sequencing of C-terminal peptides indicate that three C-terminal amino acids present in the precursor form are removed in the intermediate and in the fatty acid acylated forms. C-Terminal proteolysis appears to accompany methyl esterification, since the methylation occurs with the intermediate and the fatty acid-acylated forms, but not with the precursor. Palmitic acid is identified as the major fatty acid attached to the fatty acid-acylated form.     

COOH-terminal methylation of lamin B and inhibition of methylation by farnesylated peptides corresponding to lamin B and other CAAX motif proteins.

Previous reports from this laboratory have demonstrated that lamin B is reversibly methylesterified in a cell cycle-dependent manner. The site of this methylation, however, was not identified. In this report, we describe a single major methylated product obtained following reversed-phase high-performance liquid chromatographic analysis of peptides generated by proteolytic digestion of lamin B from rat liver nuclear envelopes. This peptide was retained on a lamin B COOH-terminal-specific antibody-affinity column, and COOH-terminal localization was confirmed by amino acid sequencing. Two other COOH-terminal peptides were found but were not methylated and differed in sequence by at least a single residue from the methylated peptide, indicating the existence of two lamin B gene products. Tetrapeptides, representing the putative mature COOH termini of lamin B, K-ras-2A, and unprocessed lamin A, were synthesized with or without farnesyl modification of the COOH-terminal cysteines. All three farnesylated peptides served as substrates for the partially purified lamin B methyltransferase with apparent Km values of 4.5, 0.69, and 21 microM, respectively. Nonfarnesylated peptides were not substrates for the enzyme. The three farnesylated peptides were also effective to varying degrees at inhibiting the methylation of lamin B and other cellular proteins in cell lysates.     

N-glycohydrolysis of adenosine diphosphoribosyl arginine linkages by dinitrogenase reductase activating glycohydrolase (activating enzyme) from Rhodospirillum rubrum

The reaction catalyzed by the activating enzyme for dinitrogenase reductase from Rhodospirillum rubrum has been studied using an ADP-ribosyl hexapeptide, obtained from proteolysis of inactive dinitrogenase reductase, and synthetic analogs such as N alpha-dansyl-N omega-ADP-ribosylarginine methyl ester. The activating enzyme catalyzed N-glycohydrolysis of the ribosyl-guanidinium linkage releasing ADP-ribose and regenerating an unmodified arginyl guanidinium group. Optimal glycohydrolysis of the low molecular weight substrates occurred at pH 6.6 and required 1 mM MnCl2, but did not require ATP. The ADP-ribosyl hexapeptide (Km 11 microM), N alpha-dansyl-N omega-ADP-ribosylarginine methyl ester (Km 12 microM), N alpha-dansyl-N omega-ADP-ribosylarginine (Km 12 microM), N alpha-dansyl-N omega-1,N6-etheno-ADP-ribosylarginine methyl ester (Km 11 microM), and N alpha-dansyl-N omega-GDP-ribosylarginine methyl ester (Km 11 microM) were comparable substrates. N omega-ADP-ribosylarginine (Km 2 mM) was a poor substrate, and the activating enzyme did not catalyze N-glycohydrolysis of N alpha-dansyl-N omega-5'-phosphoribosylarginine methyl ester or N alpha-dansyl-N omega-ribosylarginine methyl ester. 13C NMR of N alpha-tosyl-N omega-ADP-ribosylarginine methyl ester established that the activating enzyme specifically hydrolyzed the alpha-ribosyl-guanidinium linkage. The beta-linked anomer was hydrolyzed only after anomerization to the alpha configuration. We recommend [arginine(N omega-ADP-alpha-ribose)]dinitrogenase reductase N-glycohydrolase (dinitrogenase reductase activating) and dinitrogenase reductase activating glycohydrolase as the systematic and working names for the activating enzyme.     

Oxidation and oxygenation of L-amino acids catalyzed by a L-phenylalanine oxidase (deaminating and decarboxylating) from Pseudomonas sp. P-501

A number of L-amino acids and derivatives were tested as substrates for the purified Pseudomonas L-phenylalanine oxidase. The reaction products of these amino acids were analyzed by high performance liquid chromatography and the kinetic properties of the reactions were partially characterized. In addition to L-phenylalanine, L-tyrosine, DL-o-tyrosine, DL-m-tyrosine, p-fluoro-DL-phenylalanine and beta-2-thienyl-DL-alanine served as substrates for both oxidation and oxygenation catalyzed by the enzyme. On the other hand, L-methionine and L-norleucine were enzymically converted to the corresponding alpha-keto acids with the consumption of oxygen and with the formation of ammonia and hydrogen peroxide in stoichiometric amounts. Kinetic studies showed that the Km values for oxidation and oxygenation of L-phenylalanine by the enzyme were 2.04 mM and 1.96 mM for oxygen, and 13.3 microM and 11.1 microM for L-phenylalanine, respectively. omega-Phenyl fatty acids such as phenylacetic acid, 3-phenylpropionic acid and 4-phenylbutyric acid were competitive inhibitors of the enzyme towards L-phenylalanine. Both oxidation and oxygenation of L-phenylalanine by the enzyme were also inhibited by phenylacetic acid competitively.     

Identification of an autoinhibitory domain in the C-terminal region of the plant plasma membrane H(+)-ATPase.

Proteolytic (trypsin) treatment removes a small terminal segment from the 100-kDa plant plasma membrane H(+)-ATPase. This results in activation of H+ pumping across the plasma membrane, suggesting that an inhibitory domain is located in one of the terminal regions of the enzyme (Palmgren, M.G., Larsson, C., and Sommarin, M. (1990) J. Biol. Chem. 265, 13423-13426). In order to identify the origin of the fragment released by trypsin, polyclonal antibodies were raised against the first 55 amino acids (N-terminal region), the last 99 amino acids (C-terminal region), and a portion of 150 amino acids in the central part of the enzyme as deduced from one of the H(+)-ATPase genes (PMA2) of Arabidopsis thaliana. The native, 100-kDa H(+)-ATPase was recognized by all three antisera in Western blots. By contrast, the approximately 90-kDa polypeptide appearing after trypsin treatment was only recognized by the antisera against the N-terminal and central region, but not by the antiserum against the C-terminal region, suggesting that the inhibitory domain is located in this part of the enzyme. To more closely determine the position of the inhibitory domain, three peptides representing conserved parts of the C-terminal region were synthesized (residues 861-888, 912-943, and 936-949 of the Arabidopsis (PMA2) sequence). Only one of the peptides (residues 861-888) affected H+ pumping by the trypsin-activated (approximately 90-kDa) enzyme. This peptide of 28 amino acids inhibited H+ pumping with an IC50 of about 15 microM, suggesting that the auto-inhibitory domain is located within the corresponding part of the C-terminal region.     

Schistosoma mansoni: purification and characterization of the major acidic proteinase from adult worms

We report purification of the major digestive proteinase from adult worms of Schistosoma mansoni. This enzyme is a thiol proteinase with a pH optimum of 5 and is activated by thiol reagents. It was purified 300-fold using a combination of gel chromatography and chromatofocusing. It readily hydrolyzed hemoglobin with an apparent Km of 0.29 microM and a specific activity of 27 micrograms degraded/min/mg enzyme at 37 C. Peptides with positively charged amino acids were preferentially cleaved. The enzyme degraded Boc-Arg-Arg-7-amino-4-methyl coumarin with a kcat/Km of 9083 M-1 sec-1. Lengthening the peptide chain to 3 amino acids or substituting glycine for the amino terminal arginine resulted in decreased activity. The enzyme was inhibited by chloromethylketone-derivatized peptides of similar sequence and by leupeptin. The purified proteinase exhibits microheterogeneity in different preparations with forms ranging in molecular weight from 30,000 to 35,000, and pI 5.7-6.0.     

Purification and characterization of rat skeletal muscle fructose-6-phosphate,2-kinase:fructose-2,6-bisphosphatase

Fructose-6-phosphate,2-kinase:fructose-2,6-bis-phosphatase from rat skeletal muscle has been purified to homogeneity, and its structure and kinetic properties have been determined. The Mr of the native enzyme was 100,000 and the subunit Mr was 54,000. The apparent Km values of fructose-6-P,2-kinase for Fru-6-P and ATP were 56 and 48 microM, respectively. The apparent Km value for Fru-2,6-P2 of fructose-2,6-bis-phosphatase was 0.4 microM, and the Ki for Fru-6-P was 12.5 microM. The enzyme was bifunctional, and the phosphatase activity was 2.5 times higher than the kinase activity. The enzyme was not phosphorylated by cAMP-dependent protein kinase. The amino acid composition of the skeletal muscle enzyme was similar to that of the rat liver enzyme, and the carboxyl terminus sequence (His-Tyr) was the same as that of the liver enzyme. The tryptic peptides generated from the liver and skeletal muscle enzymes were identical except for two peptides. A peptide corresponding to nucleotides 14-28 of the rat liver enzyme was not detected in the skeletal muscle enzyme. A peptide whose amino acid sequence was Thr-Ala-Ser-Ile-Pro-Gln-Phe-Thr-Asn-Ser-Pro-Thr-Met-Val-Ile-Met-Val-Gly-Leu-Pro - Ala-Arg was also isolated. This peptide was the same as that of rat liver enzyme (nucleotides 31-52) containing the phosphorylation site except in the muscle enzyme two amino terminus amino acids, Gly-Ser(P), have been altered to Thr-Ala. Thus, the rat skeletal muscle enzyme is very similar in structure to the rat liver enzyme except for the lack of possibly one peptide and the lack of a phosphorylation site by the substitution of the target Ser with Ala.     

Inhibition of the action of the stimulatory GDP/GTP exchange protein for smg p21 by the geranylgeranylated synthetic peptides designed from its C-terminal region

smg p21B, a member of the ras p21-like small GTP-binding protein superfamily, undergoes post-translational modifications, which are geranylgeranylation of the cysteine residue in the C-terminal region followed by removal of the three C-terminal amino acids (QLL) and the subsequent carboxyl methylation of the exposed prenylated cysteine residue. smg p21B has a polybasic region upstream of the prenylated cysteine residue. We have previously proposed that these C-terminal structures of smg p21B are essential for the action of its stimulatory GDP/GTP exchange protein, named GDP dissociation stimulator (GDS). We studied here which structure of the C-terminal region of smg p21B is important for its interaction with smg p21 GDS. For this purpose, we synthesized a peptide according to the C-terminal structure of smg p21B, which was PGKARKKSSC-geranylgeranyl-carboxyl methyl, and its variously modified peptides and examined their ability to interact with smg p21 GDS and to interfere with the smg p21 GDS action to stimulate the GDP/GTP exchange reaction of smg p21B. The results indicate that the phosphorylated form of PGKARKKSSC-geranylgeranyl stoichiometrically interacts with smg p21 GDS, that the presence of the geranylgeranyl moiety is essential for, but not sufficient for, the smg p21 GDS action, and that the presence of the methyl moiety, removal of the three C-terminal amino acids, and the presence of the polybasic amino acids also affect the smg p21 GDS action. It is likely that all the steps of the post-translational processing and presence of the polybasic region in the C-terminal region of smg p21B are related to its interaction with smg p21 GDS.     

Synthetic fragments of beta-casein as model substrates for liver and mammary gland casein kinases

The octapeptide Glu-Ser-Leu-Ser-Ser-Ser-Glu-Glu, corresponding to the 14-21 sequence of bovine beta-casein A2 and 11 shorter and/or modified derivatives were synthesized and used as model substrates for three casein kinases: rat liver casein kinases 2 and 1 and a casein kinase isolated from the golgi-enriched fraction of lactating mammary gland (GEF-casein kinase). Casein kinase-2 readily phosphorylates the octapeptide at its Ser-4 residue with a Vmax value comparable to those obtained with protein substrates and Km values of 85 microM and 11 microM in the absence and presence of polylysine, respectively. These are the most favourable kinetic parameters reported so far with peptide substrates of casein kinase-2. Stepwise shortening of the octapeptide from its N terminus promotes both a gradual decrease of Vmax and an increase of Km, this being especially dramatic in passing from the hexapeptide Leu-Ser-Ser-Ser-Glu-Glu (Km 210 microM) to the pentapeptide Ser-Ser-Ser-Glu-Glu (Km 2630 microM). The tetrapeptide Ser-Ser-Glu-Glu is the shortest derivative still phosphorylated by casein kinase-2, albeit very slowly, and the tripeptides Ser-Glu-Glu and Glu-Leu-Ser were not substrates at all. Furthermore, the pentapeptide Ser-Ser-Ser-Glu-Glu was found to be a better substrate than Ser-Ser-Ala-Glu-Glu, Ser-Ala-Ser-Glu-Glu and Ser-Ala-Ala-Glu-Glu by virtue of its lower Km value. These data, while confirming that the motif Ser-Xaa-Xaa-Glu is specifically recognized by casein kinase-2, strongly suggest that additional local structural features can improve the phosphorylation efficiency of serine-containing peptides which are devoid of the large acidic clusters recurrent in many phosphorylation sites of casein kinase 2. In particular, predictive structural analysis as well as NMR and C18 reverse-phase HPLC elution profile data support the hypothesis that a beta-turn conformation is responsible for the remarkable suitability of the octapeptide Glu-Ser-Leu-Ser-Ser-Ser-Glu-Glu and some of its shorter derivatives to phosphorylation mediated by casein kinase-2. While neither the peptide Glu-Ser-Leu-Ser-Ser-Ser-Glu-Glu nor any of its derivatives were affected by casein kinase-1, a rapid phosphorylation of the octapeptide by GEF-casein kinase at Ser-5 (not Ser-4) was obtained.(ABSTRACT TRUNCATED AT 400 WORDS)     

S-farnesylation and methyl esterification of C-terminal domain of yeast RAS2 protein prior to fatty acid acylation

Posttranslational processing/modification is required for membrane localization and activation of ras proteins. In the case of yeast RAS2 protein, we have reported that the process starts with the removal of the initiator methionine followed by polyisoprenylation, removal of 3 amino acid residues from the C terminus, methyl esterification, and fatty acid acylation (Fujiyama, A., and Tamanoi, F. (1990) J. Biol. Chem. 265, 3362-3368). In this study, we demonstrate that polyisoprenylation and methyl esterification of the cysteine residue in the C-terminal domain of the RAS2 protein are involved in the conversion process from precursor form to intermediate form. The polyisoprenoid moiety attached to the RAS2 protein was identified as a 15-carbon farnesyl group through two independent experiments: the release of S-farnesylcysteine with carboxypeptidase Y from the RAS2 protein, and the recovery of radioactive farnesol through methyliodide treatment of the RAS2 protein purified from yeast cells labeled with [3H]mevalonic acid. The farnesyl group attached to the RAS2 protein was detected predominantly in the C-terminal peptide, SGSGGCC, both in the intermediate and in the fatty acid acylated RAS2 protein. The C-terminal cysteine of the intermediate protein is also modified by methyl esterification in a nearly stoichiometric manner.     

Interactions of alpha-chymotrypsin and Carlsberg subtilisin with methyl N alpha-acetyl-2-(alkylthio)-L-tryptophanoates

Methyl N alpha-acetyl-2-(alkylthio)-L-tryptophanoates bearing different alkylthio groups were synthesized and employed as substrates for alpha-chymotrypsin and Carlsberg subtilisin in an attempt to investigate the properties of the hydrophobic pocket or cleft (S1 subsite) of the enzymes which accommodates the side-chain of the P1 amino acid residue of the substrates. The derivatives with ethylthio, 2-hydroxyethylthio, 2,3-dihydroxypropylthio, 2-aminoethylthio, carboxymethylthio, 2-carboxyethylthio, 1,2-dicarboxyethylthio, and 2-amino-2-carboxyethylthio (cysteinyl-S) groups were hydrolyzed by alpha-chymotrypsin but with kcat/Km values 4.6 to 15 times smaller than that of methyl N alpha-acetyl-L-tryptophanoate, due mainly to larger Km values. The glutathionyl derivative was only weakly bound to the enzyme. Analyses of the kinetic parameters suggested that the S1 pocket of alpha-chymotrypsin is rather more spacious than has been supposed and is able to interact flexibly with substrates so as to orient the scissile bond to the catalytic residues. On the other hand, none of the derivatives were hydrolyzed by Carlsberg subtilisin but they all inhibited the enzyme with Ki values which are generally smaller than the Km values for N alpha-acetyl-L-aromatic (modified aromatic) amino acid methyl esters. The S1 cleft of Carlsberg subtilisin interacts rather strongly with the derivatives but lacks the flexibility necessary for catalysis.