Isopropylmalate

The leucme-reversible toxicity of 5,5, -trifluoroleudne (10) to microorganisms, such as Escherichia coli, has been attributed to incorporation of JO into protem [32] and to the action of 10 as a false feedback inhibitor of a-isopropylmalate... 

Yeast isopropylmalate isomerase of the leucine biosynthetic pathway, which catalyzes a totally analogous reaction to that of aconitase, converts 3-hydroxy-3-carboxy-4-methylpentanoate to 2-hydroxy-3-carboxy-4-methylpentanoate via an allylic intermediate. In its initial characterization by EPR spectroscopy, a high-field shift in its EPR signal from a g-average of 1.96 to 1.90 is seen upon addition of substrate (70). This result suggests that its mechanism is the same as that found for aconitase. 

This enzyme [EC 4.2.1.33], also referred to as isopropyl-malate hydro-lyase, catalyzes the conversion of 3-isopro-pylmalate to 2-isopropylmaleate and water. 

ISOPROPYLMALATE DEHYDRATASE 3-ISOPROPYLMALATE DEHYDROGENASE 2-ISOPROPYLMALATE SYNTHASE ISOPULLULANASE ISOSBESTIC POINT... 

Other enzymes in the aconitase family include isopropylmalate isomerase and homoaconitase enzymes functioning in the chain elongation pathways to leucine and lysine, both of which are pictured in Fig. 17-18.90 There are also iron-sulfur dehydratases, some of which may function by a mechanism similar to that of aconitase. Among these are the two fumarate hydratases, fumarases A and B, which are formed in place of fumarase C by cells of E. coli growing anaerobically.9192 Also related may be bacterial L-serine and L-threonine dehydratases. These function without the coenzyme pyridoxal phosphate (Chapter 14) but contain iron-sulfur centers.93-95 A lactyl-CoA... 

Another new alkaloid (50) was also isolated from P. heterophylla. Mild acid hydrolysis of (50) gave a desacetyl compound which showed a prominent peak due to the loss of —CH2C02CH3 in its mass spectrum, thereby indicating that the other carboxyl group is esterified to the base. Vigorous acid hydrolysis of (50) gave 1-hydroxymethylpyrrolizidine of undefined stereochemistry together with a neutral compound which yielded (-)-2-isopropylmalic acid on basic hydrolysis. 

Isopropylmalate isomerase (Leu biosynthesis) a-Isopropyl malate/a-hydroxy-P-carboxy-i socaprate... 

Release of water from Fea, H bridge of the serine base to —H (Ser642 is conserved in aconitase and in isopropylmalate isomerase). 

Figure 4 Analogous substrates of hydrolytic Fe/S enzymes, (a) Citrate (R = acetyl), isopropylmalate (R = propyl), malate (R = H) (b) serine (c) dihydroxyvalerate (R = methyl), dihydroxymethylvalerate (R = ethyl).

The substrates isocitrate and isopropylmalate are structurally similar, i.e., OOC(HO)CHCH(X)COOa where X represents the y-moiety the —CH2COO of isocitrate and the -CH(CH3)2 of isopropylmalate (Fig. 5). Coenzyme NADP differs from NAD only by a phosphate group esterified at the 2 C of the adenosine ribose. Presumably, distinct functions of NAD-IDH, NADP-IDH, and NAD-IMDH are conferred by their ability to recognize alternative substrates and coen-... 

Figure 5 Structures of 2R,35-isocitrate (A), 2/ ,3S-isopropylmalate (B), and 2R,3S-homoisocitrate (C). a, P refers to the a- and fi-carboxyl groups, respectively. The unique y-moieties are presented in bold.

Figure 6 Schematic diagram of the active site of the E. coli NADP-IDH with bound 2A,3>S-isocitrate (A) and the active site of T. thermophilus NAD-IMDH with bound 2R,3S-isopropylmalate (B). A prime indicates the residues donated from the second subunit, a, 3 refers to a- and P-carboxyl groups, respectively. The major determinants of substrate specificity are residues Seri 13 and Asnll5 in the NADP-IDH, and Glu87 and Leu90 in the NAD-IMDH, which interact with the unique y-moieties of the substrates. (Modified from Ref. 15.)...

K Imada, M Sato, N Tanaka, YKatsube, Y Matsuura, T Oshima. Three-dimensional structure of a highly thermostable enzyme, 3-isopropylmalate dehydrogenase of Thermus thermophilus at 2.2 A resolution. J Mol Biol 222 725-738, 1991. 

RD Chen, A Greer, JM Hurley, AM Dean. Engineering secondary structure to invert coenzyme specificity in isopropylmalate dehydrogenase. In DR Marshak, ed. Techniques in Protein Chemistry VIII. New York Academic Press, 1997, pp 809-816. 

AM Dean, L Dvorak. The role of glutamate 87 in the kinetic mechanism of Thermus thermophilus isopropylmalate dehydrogenase. Protein Sci 4 2156-2167, 1995. 

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