Yeast synthetase

It has been reported that, like liver acetyl-CoA carboxylase, both the liver and yeast fatty acid synthetases are inhibited by low concentrations (0.5 to 5 X 10 71/) of long-chain fatty acyl-CoA derivatives, the longer-chain derivative producing greater inhibition [226,246,247]. In the case of the yeast synthetase, inhibition by long-chain acyl-CoA derivatives was competitive with respect to acetyl-CoA and NADPH. For the same reasons alluded to earlier in the discussion of the inhibition of acetyl-CoA carboxylase by fatty acyl-CoA derivatives, some caution must be exercised in interpreting the effect of these potent inhibitors (see Section V, C, 2). 

Fatty acid synthetases can be divided mainly into Type I and Type II enzymes (Table 3.8). Type I synthetases are multifunctional proteins in which the proteins catalysing the individual partial reactions are discrete domains. This type includes the animal synthetases and those from higher bacteria and yeast. Type II synthetases contain enzymes which can be separated, purified and studied individually. This system occurs in lower bacteria and plants and has been studied most extensively in E. coli. In addition, Type III synthetases - occurring in different organisms - catalyse the addition of C2 units to preformed acyl chains and are also known as elongases. Although, historically, the reactions of the yeast synthetase were unravelled first, we shall start by describing the separate reactions catalysed by the enzymes of E. coli. 

Basically, there are two completely different pathways by which unsaturated fatty acids are produced. In an earlier section, we mentioned that the fatty acid synthetase of E, coli, in contrast to the mammalian and yeast synthetases, produced unsaturated as well as saturated acids. An idea of... 

In the organism tissues, fatty acids are continually renewed in order to provide not only for the energy requirements, but also for the synthesis of multicomponent lipids (triacylglycerides, phospholipids, etc.). In the organism cells, fatty acids are resynthetized from simpler compounds through the aid of a supramolecular multienzyme complex referred to as fatty acid synthetase. At the Lynen laboratory, this synthetase was first isolated from yeast and then from the liver of birds and mammals. Since in mammals palmitic acid in this process is a major product, this multienzyme complex is also called palmitate synthetase. 

The formation of 0-seryl or 0-prolyl esters (Figure 1) of certain N-hydroxy arylamines has been inferred from the observations that highly reactive intermediates can be generated in vitro by incubation with ATP, serine or proline, and the corresponding aminoacyl tRNA synthetases (11,12,119). For example, activation of N-hydroxy-4-aminoquinoline-l-oxide (119,120), N-hydroxy-4-aminoazobenzene (11) and N-hydroxy-Trp-P-2 (121) to nucleic acid-bound products was demonstrated using seryl-tRNA synthetase from yeast or rat ascites hepatoma cells. More recently, hepatic cytosolic prolyl-, but not seryl-, tRNA synthetase was shown to activate N-hydroxy-Trp-P-2 (12) however, no activation was detectable for the N-hydroxy metabolites of AF, 3,2 -dimethyl-4-aminobiphenyl, or N -acetylbenzidine (122). 

SMM synthesis is mediated by the enzyme methionine S-methyltransferase (MMT) through the essentially irreversible, AdoMet-mediated methylation of methionine.48"5 Both MMT and SMM are unique to plants 48,50 The opposite reaction, in which SMM is used to methylate homocysteine to yield two molecules of methionine, is catalyzed by the enzyme homocysteine S-methyltransferase (HMT).48 Unlike MMT, HMTs also occur in bacteria, yeast, and mammals, enabling them to catabolize SMM of plant origin, and providing an alternative to the methionine synthase reaction as a means to methylate homocysteine. Plant MMT and HMT reactions, together with those catalyzed by AdoMet synthetase and AdoHcy hydrolase, constitute the SMM cycle (Fig. 2.4).4... 

This selection scheme was used to evolve the orthogonal E. coli tRNA u -TyrRS pair in yeast. A synthetase library (10 in size) was similarly constructed by randomizing five active-site residues in E. coli TyrRS corresponding to the five residues randomized in the Af/TyrRS. Mutant synthetases were identified after several rounds of positive and negative selection that incorporate a number of unnatural amino acids into proteins, albeit with rather low protein yields (about 0.05 mgl A similar approach has been used to evolve orthogonal E. coli leucyl tRNAcuA LeuRS pairs that selectively incorporate photochromic and fluorescent amino acids into proteins in yeast. ... 

About 10-25%, i.e. 50-200 pg, of the daily dietary intake of folic acid in yeasts, liver, and green vegetables is absorbed via active and passive transport in the proximal jejunum. As humans do not have dihydropteroate synthetase, which synthesizes folic acid in bacteria, we require folic acid in the diet. Only small amounts of folate can be stored in the body and dietary deficiency for only a few days can result in symptomatic folate deficiency. 

Further detailed study of the substrate specificity of yeast squalene synthetase has been reported (see Vol. 7, p. 130). The enzyme is very sensitive to changes in substrate. For example, 10,11-dihydrofarnesyl pyrophosphate was converted into 2,3,22,23-tetrahydrosqualene with only 60% of the efficiency of farnesyl pyrophosphate whereas 6,7-dihydro- and 6,7,10,11-tetrahydro-farnesyl pyrophosphates were not metabolized. The first of the two binding sites has a greater preference for farnesyl pyrophosphate and this accounts for the formation of the unsymmetrical squalene product when mixtures of farnesyl pyrophosphate and a modified substrate are used. The importance of the methyl groups, especially that at C-3, for binding is emphasized by the low efficiency of conversion of 3-desmethylfarnesyl, , -3-methylundeca-2,6-dien-l-yl (1), and E,E-7-desmethylfarnesyl pyrophosphates. The prenylated cyclobutanones (2) and (3)... 

The results of these efforts show that no method of tRNA recognition is universal.2443 In some cases, e.g., for methionine- or valine-specific tRNAs, the synthetase does not aminoacylate a modified tRNA if the anticodon structure is incorrect. Although the anticodon is 7.5 ran away from the CCA end of the tRNA, the synthetases are large enzymes. Many of them are able to accommodate this large distance between a recognition site and the active site (Fig. 29-9A). For some other tRNAs the anticodon is not involved in recognition 245 For yeast tRNAphe residues in the stem of the dihydrouridine loop and at the upper end of the amino acid acceptor stem seem to be critical.241... 

Mechanisms of reaction. Activation of an amino acid occurs by a direct in-line nucleophilic displacement by a carboxylate oxygen atom of the amino acid on the a phosphorus atom of MgATP to form the aminoacyl adenylate (Eq. 29-1, step a). For yeast phenylalanyl-tRNA synthetases the preferred form of MgATP appears to be the P,y-bidentate (A screw sense) complex (p. 643).250 This is followed by a second nucleophilic displacement, this one on the C = 0 group of the aminoacyl adenylate by the -OH group of the tRNA (Eq. 29-1, step b Fig. 29-9C). A conformational change in the protein may be required to permit dissociation of the product, the aminoacyl-tRNA. In the complex of a class I synthetase with aminoacyl... 

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