Substrate binding domain surrogates

The goal of the present study was to develop a computer-based cubic section model of the substrate binding domain of HLADH. It was considered that the Jones cubic section model could be refined by use of computer assisted substrate overlay in combination with kinetic data on a wide variety of substrates. As in the Jones approach we used the alcohol products as the surrogate substrate structures. Thus, we determined the low energy conformation of alcohols produced from ketones that have been reported to be reduced by HLADH and for which comparative kinetic data vs cyclohexanol could be calculated. As well, we determined the preferred conformations of all alcohols that would have been produced from ketones subjected to but failing to undergo HLADH reduction. These calculations utilised molecular mechanics (MACROMODEL) and yielded accurate co-ordinates for ali atoms in each alcohol. Where enantiomeric or stereoisomeric alcohols were produced or capable of production, the co-ordinates of each were calculated. 

The X-ray crystallographic structure of the ternary complex, HLADH-NADH-dimethyl sulfoxide, DMSO, has been determined to 2.9 The crystallographic co-ordinates available from the Brookhaven protein data bank were entered into MACROMODEL and the amino acid residues surrounding the active site identified. Using MACROMODEL and ENZYME the substrate binding domain of HLADH was mapped as a cubic section model. The model obtained from this approach were compared with model col 3 obtained by substrate surrogate overlay. 

Model col 3 and the model constructed with all hydrogens in substrate surrogates (co13h) were compared with the cubic section model derived from display of the cubes occupied by residues of HLADH lining the substrate binding domain. Using... 

Comparison of the X-ray based cubic section model with the substrate surrogate derived cubic section model gave a view of the amino acid residues around the substrate binding domain (Figure 21). Forbidden and boundary regions defined by the substrate surrogate overlay method are near the side chains amino acid residues. Substrate interactions with surface of the amino acid residues could be estimated by molecular mechanics (MACROMODEL). 

Figure 3. Three of the modules comprising methionine synthase. At the top center is the Bi2- binding fragment [651-896], a structure with two domains, one a four-helix bundle that serves to cap the cofactor, and the other an a/p fold that interacts with the lower face of the corrin macrocycle and binds the nucleotide tail of cobalamin. Measurements of the rates of photolysis of the C0-CH3 bond indicate that the cap domain covers the upper face of the corrin in the substrate-free form of the intact enzyme (7). On the lower right is the activation domain [897-1227] with bound AdoMet. This helmet-shaped single domain is an unusual fold with no resemblance to other well-characterized AdoMet-binding domains (8). On the lower left is the structure of the methyltransferase AcsE from Moorella thermoaceticum, which we take as a surrogate for the folate-binding domain of MetH.

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