Showing papers by "Steven Clarke published in 2001"

Neighboring side chain effects on asparaginyl and aspartyl degradation: an ab initio study of the relationship between peptide conformation and backbone NH acidity.

Abstract: The rate of spontaneous degradations of asparagine and aspartyl residues occurring through succinimide intermediates is dependent upon the nature of the residue on the carboxyl side in peptides. For nonglycine residues, we show here that this effect can largely be attributed to the electrostatic/inductive effect of the side chain group on the equilibrium concentration of the anionic form of the peptide bond nitrogen atom that initiates the succinimide forming reaction. However, the rate of degradation of Asn-Gly and Asp-Gly containing peptides is about an order of magnitude greater than predicted solely using this explanation. To understand the nature of the glycine effect, ab initio calculations were performed on model compounds. These calculations indicate that there is little to no change in the stability of the transition state or the tetrahedral intermediate of succinimide formation with Asn-/Asp-Gly and Asn-/Asp-Ala derivatives. However, we have found that the acidity of the backbone peptide nitrogen NH is highly dependent upon the conformation of the molecule. Since glycine residues lack the beta-carbon common to all other protein amino acids, these residues can sample additional regions of conformational space where it is possible to further stabilize the backbone amide anion and thus increase the rate of degradation. These results provide the first rationale for the particular rate enhancement of degradation in peptidyl Asn-/Asp-Gly sequences. The results also can be applied to asparagine and aspartyl residues in proteins where the 3-dimensional structure provides additional constraints on conformation that can either increase or decrease the equilibrium concentration of the backbone amide anion and thus their rate of degradation via succinimide intermediates. Understanding this chemistry will assist attempts to minimize the deleterious effect of aging at the molecular level. The relationship between these results and proton exchange experiments is discussed in the Appendix.

7 Postisoprenylation protein processing: CXXX (CaaX) endoproteases and isoprenylcysteine carboxyl methyltransferase

Abstract: Publisher Summary This chapter discusses the progress in understanding the “postisoprenylation” processing of CXXX proteins—the endoproteolytic processing step and the carboxyl methylation step. For much of the 1990s, getting a handle on these steps was slow, at least when compared with the rapid progress in understanding the protein isoprenyltransferases, and there was uncertainty about their physiologic importance. More recently, however, there have been exciting advances in understanding these steps, making it an attractive time to review this area. In a landmark study, Boyartchuk, Ashby, and Rine identified two genes from Saccharomyces cerevisiae , RCEl and AFCl, that are involved in the proteolytic removal of the “-XXX” from two farnesylated CXXX proteins (Ras2p and the precursor to the yeast mating pheromone a-factor). This breakthrough made it possible to mine the expressed sequence tag (EST) databases and clone the mammalian orthologs for RCEl and AFCl. The chapter provides an overview of earlier work describing the endoproteolysis and carboxyl methylation enzymatic activities, as well as newer work on the identification of the gene products responsible for the endoproteolysis and carboxyl methylation steps in yeast and mammals.

Distinct Patterns of Expression But Similar Biochemical Properties of Protein l-Isoaspartyl Methyltransferase in Higher Plants

Abstract: Protein L-isoaspartyl methyltransferase is a widely distributed repair enzyme that initiates the conversion of abnormal L-isoaspartyl residues to their normal L-aspartyl forms. Here we show that this activity is expressed in developing corn (Zea mays) and carrot (Daucus carota var. Danvers Half Long) plants in patterns distinct from those previously seen in winter wheat (Triticum aestivum cv Augusta) and thale cress (Arabidopsis thaliana), whereas the pattern of expression observed in rice (Oryza sativa) is similar to that of winter wheat. Although high levels of activity are found in the seeds of all of these plants, relatively high levels of activity in vegetative tissues are only found in corn and carrot. The activity in leaves was found to decrease with aging, an unexpected finding given the postulated role of this enzyme in repairing age-damaged proteins. In contrast with the situation in wheat and Arabidopsis, we found that osmotic or salt stress could increase the methyltransferase activity in newly germinated seeds (but not in seeds or seedlings), whereas abscisic acid had no effect. We found that the corn, rice, and carrot enzymes have comparable affinity for methyl-accepting substrates and similar optimal temperatures for activity of 45 degrees C to 55 degrees C as the wheat and Arabidopsis enzymes. These experiments suggest that this enzyme may have specific roles in different plant tissues despite a common catalytic function.

Distinct reactions catalyzed by bacterial and yeast trans-aconitate methyltransferases.

Abstract: The trans-aconitate methyltransferase from the bacterium Escherichia coli catalyzes the monomethyl esterification of trans-aconitate and related compounds. Using two-dimensional 1 H/ 13 C nuclear magnetic resonance spectroscopy, we show that the methylation is specific to one of the three carboxyl groups and further demonstrate that the product is the 6-methyl ester of trans-aconitate (E-3-carboxy-2- pentenedioate 6-methyl ester). A similar enzymatic activity is present in the yeast Saccharomyces cereVisiae. Although we find that yeast trans-aconitate methyltransferase also catalyzes the monomethyl esterification of trans-aconitate, we identify that the methylation product of yeast is the 5-methyl ester ( E-3-carboxyl- 2-pentenedioate 5-methyl ester). The difference in the reaction catalyzed by the two enzymes may explain why a close homologue of the E. coli methyltransferase gene is not found in the yeast genome and furthermore suggests that these two enzymes may play distinct roles. However, we demonstrate here that the conversion of trans-aconitate to each of these products can mitigate its inhibitory effect on aconitase, a key enzyme of the citric acid cycle, suggesting that these methyltransferases may achieve the same physiological function with distinct chemistries. The conversion of citrate to isocitrate catalyzed by aconitase in the tricarboxylic acid cycle of almost all organisms involves the intermediate formation of cis- aconitate. This compound is generally not released from the enzyme complex, but when it is, it can rebind and undergo the transformation into isocitrate ( 1). cis-Aconitate is not chemically stable and is readily converted to the more stable trans-aconitate form (2-5), which has been shown to be an effective inhibitor of aconitase from a number of organisms (6-10). It is unclear how cells prevent the accumulation of this potentially toxic metabolite. We previously identified a novel methyltransferase that esterifies trans-aconitate in Escherichia coli and have shown that it is encoded by the tam gene (11). This activity has also been detected in the yeast Saccharomyces cereVisiae although its structural gene was not identified ( 11). These activities can potentially result in the further metabolism of trans-aconitate, either by forming noninhibitory complexes or by potentially catalyzing one step in the conversion back to cis-aconitate. To begin to understand the metabolism of trans-aconitate by the methylation pathway, we first focused on determining the exact products of the reactions. It had not been established which of the three distinct carboxyl groups are modified and whether other covalent changes, such as a possible trans-cis isomerization may also ac- company the enzymatic reaction. trans-Aconitate can be potentially methyl-esterified to make one trimethyl, three structurally distinct dimethyl, and three structurally distinct monomethyl esters of trans- aconitate. We previously determined that the E. coli Tam 1 enzyme catalyzes the formation of monomethyl ester(s) by mass spectrometry (11). Enzymatically methylated trans- aconitate was found to coelute with only one of the chemically synthesized monomethyl esters, suggesting that methylation occurs on a specific site in trans-aconitate (11). Tandem mass spectrometry did not provide useful informa- tion on which of the three carboxylic acids were methylated by Tam due to the facile cleavage of the ester bond, and the initial loss of the methyl group in the fragmentation of the molecule. In this report, we have now used an advanced NMR technique to show that E. coli Tam catalyzes the formation of the 6-methyl ester of trans-aconitate. We have combined synthetic, NMR, mass spectrometric, and chro- matographic methods to also show that the yeast enzyme monomethylates trans-aconitate but on a distinct site to form the 5-methyl ester. These results show that a trans-cis isomerization does not accompany the methylation reaction and that the function of the methyltransferase in bacterial and lower eucaryotic cells may be distinct. Significantly, we have also shown that the methylesteri- fication of trans-aconitate largely relieves the inhibition of this compound on aconitase activity in both E. coli and yeast †

Identification of the gene and characterization of the activity of the trans-aconitate methyltransferase from Saccharomyces cerevisiae.

Abstract: We have identified the yeast open reading frame YER175c as the gene encoding the trans- aconitate methyltransferase of Saccharomyces cereVisiae. Extracts of a yeast strain with a disrupted YER175c gene demonstrate a complete loss of activity toward the methyl-accepting substrates trans-aconitate, cis- aconitate, DL-isocitrate, and citrate. Reintroduction of the YER175c gene on a plasmid results in an overexpression of the activity toward each of these methyl-accepting substrates. We now designate this gene TMT1 for trans-aconitate methyltransferase. We examined the methyl-accepting substrate specificity of this enzyme in extracts from overproducing cells. We found that trans-aconitate was the best substrate with a Km of 0.66 mM. Other substrates were recognized much more poorly, including cis-aconitate with a Km of 74 mM and the decarboxylation product itaconate with a Km of 44 mM. The ratio of the maximal velocity to the Km of these substrates was only 0.24% and 0.9% that of trans-aconitate; for other substrates including citrate and other tricarboxylate and dicarboxylate derivatives, this ratio ranged from 0.0003% to 0.062% that of trans-aconitate. We then asked if any of these compounds were present endogenously in yeast extracts. We were able to identify trans-aconitate 5-methyl ester as well as additional unidentified radiolabeled products when S-adenosyl-L-(methyl- 3 H)methionine was mixed with TMT1 + extracts (but not with tmt1 - extracts), suggesting that there may be additional substrates for this enzyme. We showed that the product 5-methyl ester of trans-aconitate is not readily metabolized in yeast extracts. Finally, we demonstrated that the activity of the yeast trans-aconitate methyltransferase is localized in the cytosol and increases markedly as cells undergo the metabolic transition at the diauxic shift.