Enzymes synthetase complex

A second relevant enzyme to 5-FU metabolism is thymidylate synthetase (TYMS). If this enzyme is complexed with 5-FU metabolites along with 5,10-methylene-tetrahydrofolate, it cannot maintain a thymidine-5 -monophosphate pool required for DNA replication and repair. A tandem repeat polymorphism in the 5 -promoter region of the TYMS gene can increase enzyme expression (85,86). Tumors carrying the repeats have higher enzyme expression, resulting in lower response to chemotherapy compared to wild type (87). 

As an example of an asymmetric membrane integrated protein, the ATP synthetase complex (ATPase from Rhodospirillum Rubrum) was incorporated in liposomes of the polymerizable sulfolipid (22)24). The protein consists of a hydrophobic membrane integrated part (F0) and a water soluble moiety (Ft) carrying the catalytic site of the enzyme. The isolated ATP synthetase complex is almost completely inactive. Activity is substantially increased in the presence of a variety of amphiphiles, such as natural phospholipids and detergents. The presence of a bilayer structure is not a necessary condition for enhanced activity. Using soybean lecithin or diacetylenic sulfolipid (22) the maximal enzymatic activity is obtained at 500 lipid molecules/enzyme molecule. With soybean lecithin, the ATPase activity is increased 8-fold compared to a 5-fold increase in the presence of (22). There is a remarkable difference in ATPase activity depending on the liposome preparation technique (Fig. 41). If ATPase is incorporated in-... 

Several possible mechanisms can be envisaged to explain how Atjt may modulate the activity of the cellulose-synthetase complex. The effects of membrane fluidity on membrane-bound enzymes is well... 

S H Liaw, D Eisenberg (1994) Structural model for the reaction mechanism of glutamine synthetase, based on five crystal structures of enzyme-substtate complexes, Biochemistry 33(3) 675—681... 

The pathway from acetate to palmitic acid (actually a palmitic acid-acyl carrier protein complex) involves at least nine enzymes acetyl CoA synthetase, acetyl CoA carboxylase, and the seven enzyme fatty acid synthetase complex. We chose first to test the effect of these compounds on acetyl CoA carboxylase (ACCase) activity. There were several reasons to select ACCase as the... 

Pantothenic acid has a central role in energy-yielding metabolism as the functional moiety of coenzyme A (CoA), in the biosynthesis of fatty acids as the prosthetic group of acyl carrier protein, and through its role in CoA in the mitochondrial elongation of fatty acids the biosynthesis of steroids, porphyrins, and acetylcholine and other acyl transfer reactions, including postsynthetic acylation of proteins. Perhaps 4% of all known enzymes utilize CoA derivatives. CoA is also bound by disulfide links to protein cysteine residues in sporulating bacteria, where it may be involved with heat resistance of the spores, and in mitochondrial proteins, where it seems to be involved in the assembly of active cytochrome c oxidase and ATP synthetase complexes. 

Although fatty acid 8-oxidation is catalyzed by a series of intramitochon-drial enzymes, and the fatty acyl chain is carried by CoA, fatty acid synthesis is catalyzed by a cytosolic-multienzyme complex in which the growing fatty acyl chain is bound by thioester linkage to an enzyme-bound 4 -phosphopantetheine residue. This component of the fatty acid synthetase complex is ACP. 

Fio. 6. Activities of the cyclopeptine synthetase complex (19). (1) Anthranilic acid ade-nylyltransferase (2) L-phenylalanine adenylyltransferase (3) covalent binding of anthranilic acid (4) covalent binding of L-phenylalanine (5) methylation of enzyme-bound L-phenylalanine by 5-ade-nosyl-L-methionine (6) translocation of AI-methyl-L-phenylalanine to enzyme bound anthranilic acid (7) release of cyclopeptine by cyclization. 

The pathways diverge at phosphatidate. In the synthesis of triacylglycerols, phosphatidate is hydrolyzed by a specific phosphatase to give a diacylglycerol (DAG). This intermediate is acylated to a triacylglycerol in a reaction that is catalyzed by diglyceride acyltransferase. Both enzymes are associated in a triacylglycerol synthetase complex that is bound to the endoplasmic reticulum membrane. 

Since there are three possible ways to rearrange cyclopropylcarbinyl cation (86) of the type proposed in presqualene pyrophosphate conversion, and the unwanted cyclopropylcarbinyl (86) to allyl (87) rearrangement has been found to account for 99% of total reaction flux in model studiessqualene synthetase must exert strict regiochemical control in the catalytic steps to produce the enzymatic product squalene via the kinetically and thermodynamically unfavored (ca. 0.04 % of the total non-enzymatic flux) rearrangement process (86 82). A tight enzyme-substrate complex that imposes an energy barrier... 

If the Fatty Acid Synthetase Complex only makes palmitate where do the rest of the fatty acids come from Of course palmitate can be shortened by P-oxidation. For longer fatty acids there is a fatty acid elongation system localized on the ER. The same reactions occur as in the S)mthetase, but now have individual enzymes. Palmitate is first activated to palmitoyl-CoA. The enzymes prefer C-16 or less as... 

Figure 8.6 Change in spectroscopic characteristics with the formation of an enzyme—substrate complex Fluorescence intensity of the pyridoxal phosphate group at the active site of tryptophan synthetase changes on addition of serine and indole, the substrates.

The enzyme 2-C-methyl-D-erythritol-4-phosphate synthetase appears to catalyse a Bilik reaction (Figure 6.10) the substrate l-deoxyxylulose-5-phosphate is converted to the title compound via an intermediate aldehyde, whose carbonyl derives from C3 of the substrate. The first step is thus a Bilik reaction and the aldehyde is subsequently reduced by the enzyme using NADPH as reductant, The X-ray crystal structure of the Escherichia coli enzyme in complex with the promising antimalarial Fosmidomycin (a hydroxamic acid) reveals a bound Mn " coordinated to oxygens equivalent to the substrate carbonyl and 03. The stereochemistry and regiochemistry follow the normal Bilik course, although the crystallographers favour an alkyl shift rather than a reverse aldol-aldol mechanism. The intermediate aldehyde has been shown to be a catalytically competent intermediate. 

Brief incubation of the enzyme at 0°C with radioactive acetyl-CoA led to the formation of acyl enzyme which could be isolated by chromatography on Sephadex [24]. The enzyme-substrate complex was then reacted with acetoacetyl-CoA with the concomitant formation of HMG-CoA. Further studies indicated that the functional group on the enzyme that accepted the acetyl residue was a cysteine sulfhydryl. 4 -Phosphopantetheine is known to accept acyl residues, but it was not found in this protein. The stoichiometry for acetylation was 0.7 acetyl groups per mole of enzyme since it is a dimeric protein with apparently identical subunits, this observation is surprising. Thus, it is possible that the subunits perform different functions for example, one could be regulatory. It is interesting to note that both the thiolase and HMG-CoA synthetase utilize acyl enzyme intermediates in their catalytic mechanisms. 

Methotrexate inhibits the enzyme folate reductase at two steps (Fig. 4-12). Even though the inhibition is technically a competitive one, the enzyme binds MTX much more strongly than the natural substrate, FH2 (Table 4-5). In practical terms there is no dissociation of the enzyme-drug complex. For thymidylate synthetase to continue to produce thymidylic acid (and therefore DNA), perpetual reduction of FH2 is essential for cellular survival. The efficiency of MTX inhibition of folate reductase thus indirectly becomes the mechanism by which this drug is so cytotoxic in the S phase. In addition, there is evidence that MTX may, by binding to the tetrahydrofolate coenzyme, also inhibit the thymidylate synthetase... 

There is no final consensus on whether procyanidin biosynthesis is controlled thermodynamically or enzymatically. In either case proanthocyanidins are synthesized through sequential addition of flavan-3,4-diol units (in their reactive forms as carbocations or quinone methides) to a flavan-3-ol monomer [218]. Based on the latest findings there is some evidence that different condensation enzymes might exist which are specific for each type of flavan-3,4-diol [64] and that polymer synthesis would be subject to a very complex regulatory mechanism [63]. But so far, no enzyme synthetase systems have been isolated and enzymatic conversion of flavanols to proanthocyanidins could not be demonstrated in vitro [219]. If biosynthesis was thermodynamically controlled, the variation in proanthocyanidin composition could be explained by synthesis at different times or in different compartments [64], The hypothesis of a thermodynamically controlled biosynthesis is based on the fact that naturally and chemically synthesized procyanidin dimers occur as a mixture of 4—>8 and 4—>6 linked isomers in approximate ratios of 3-4 1 [220]. Porter [164] found analogous ratios of 4—>8 and 4—>6 linkages in proanthocyanidin polymers. 

Fir.. II. Gibbs free energies of enzyme-bound complexes of mutant tyrosyl-tRNA synthetase relative to those of wild-type enzyme. [Reprinted with permission from Ref. (/5/).)... 

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