Free Energy Requirement for Domain Movement of an Enzyme

Abstract

The induced fit movement of an enzyme molecule upon the binding of substrate (1) has been characterized for many enzymes and is essential for catalysis. Domain closure of such enzymes can drastically change their active site environment from hydrophilic to hydrophobic, and such closure allows the enzymes to undergo reactions that are difficult in the aqueous phase. In order to elucidate the detailed mechanism of a given enzyme, it is necessary to estimate quantitatively the energy required for domain movement. For the last 2 decades, molecular dynamic calculations have been used to predict the domain movement of enzymes (2, 3). Recent studies indicate that single molecule measurements may also be useful in determining the energy required for domain fluctuation (4,5). Despite these trials, it has generally proven difficult to confirm quantitatively the free energy required for domain movement of an enzyme. In this paper, we present a new method of estimating the energy required for domain movement by analyzing the reactions of two aminotransferases with a series of aliphatic ␣-amino acid substrates.Escherichia coli aspartate aminotransferase (EC 2.6.1.1) (AspAT) 1 with the bound coenzyme pyridoxal 5Ј-phosphate (PLP) reacts with an ␣-amino acid to form the pyridoxamine 5Ј-phosphate (PMP) form of the enzyme and the ␣-keto acid (6 -11) as follows.AspAT is a unique "dual substrate" enzyme that catalyzes this reaction for both acidic and hydrophobic amino acids (12, 13).In the catalytic mechanism, the catalytic group Lys 258 is commonly used for both types of substrate. Arg 386 is also frequently used for recognition of the ␣-carboxyl group of both types of substrate.With acidic substrates, AspAT undergoes a large domain movement. Arg 292 * 2 moves from the outside to the inside of the active site with its accompanying water molecules and recognizes the -carboxyl group of the substrate (6 -9). (This movement of Arg 292 * is similar to that for a hydrophobic substrate (see Fig. 3c).)With hydrophobic substrates, it is known that the catalytic efficiency increases in proportion to the side chain length of a series of straight aliphatic substrates (Cn substrates) (12,13). Surprisingly, consecutive additions of a single methylene group to the substrate (from C4 to C7) produce a constant effect on the stabilization energy of the transition state (ES ‡ ) relative to the unbound state (E ϩ S) (Refs. 12 and 13; see Fig. 5, inset). The energy contribution of one methylene group in the alkyl chain to AspAT is Ϫ0.65 kcal mol Ϫ1 for longer hydrophobic substrates with Cn, n Ն 5. Similar phenomena have been reported for many enzymes (14 -17). The energetic contribution of one methylene group in the substrate is 0.3 kcal mol Ϫ1 for horseradish peroxidase (14), -0.4 kcal mol Ϫ1 for Bacillus amyloliquefaciens subtilisin (15), Ϫ0.9 kcal mol Ϫ1 for Paracoccus denitrificans AroAT (16), and Ϫ1.5 kcal mol Ϫ1 for bovine ␣-chymotrypsin (17). This apparent uniformity of the substrate-binding site will be achieved by fluctuation of the enzyme m...

Linkage between the bacterial acid stress and stringent responses: the structure of the inducible lysine decarboxylase

“…In all cases, interaction with the substrate aldimine might require closure of the active site channel to bring them into close proximity. This effect has been observed most notably in E. coli aspartate aminotransferase where the residue R292 undergoes large movements upon substrate binding leading to formation of the closed complex (Kirsch et al , 1984; Ishijima et al , 2000).…”

Structural Insight into the Inhibition of Human Kynurenine Aminotransferase I/Glutamine Transaminase K

“…This observation indicates that only large ligands may cause this N-terminal α-helix shift. In E. coli aspartate aminotransferase, domain movement was changed dramatically from an open to a closed form by the addition of only one CH 2 to the side chain of the C4 substrate CH 3 (CH 2 )C α H(NH 3 ) + COO − 47. Taken together, these results indicate that one of the two substrates (α-ketoacid and amino acid) must be big enough to initiate the conformational change in order for the transamination reaction to happen.…”

Temperature Dependence of the Enzyme-Substrate Recognition Mechanism

“…Results and Discussion Aspartate aminotransferase from the mesophile Escherichia coli (EcAT) consists of two domains, which close upon substrate binding [1,2]. Recently, we were able to estimate the free energy required for domain movement by means of kinetic studies in a series of aliphatic substrates and corresponding crystallographic studies [3]. Upon binding of the acidic substrate, the side chain of Arg292* largely moves into the active site to create bifurcated hydrogen bonds and an electrostatic interaction with the distal carboxylate (ω-carboxylate) group of the bound acidic substrate.…”