ANALYSIS OF SUBSTRATE BINDING BY SITE-SPECIFIC MUTATION

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The objective is to develop an experimental system that will enable 1) the analysis of the effects of amino acid changes on the catalytic mechanism of a eukaryotic enzyme; 2) the demonstration of the potential of directed mutagenesis for re-engineering the properties of an enzyme and 3) the testing and refinement of the equations quantitative structure activity relations (QSAR) currently being used to describe the binding of a ligand to an enzyme. By genetically modifying an already well-studied enzyme (papain) it will be possible to test further the validity of the constants in the equations used to describe the interaction. The specific aim is to transfer the gene for the enzyme papain from the papaya plant into a plasmid of the bacterium E. coli. The sequence of nucleotides in the gene that codes for the amino acid sequence in the protein will then be determined. Using one of the techniques for site-specific mutation, the codes for the amino acid glutamine at position 142 will be changed to that which codes for glycine, or lysine. This will either remove the side chain of the glutamine (as in the change to glycine) which is thought to play a role as a backstop against which the substrate for the enzyme is lodged, or will substitute the lysine side chain that is thought to play the same role in actinidin, a similar enzyme found in Kiwi fruit. The cysteine-25 of the active site will be changed to a serine, as is found in serine proteases and will test the interchangeability of those amino acids in the enzymatic reaction. The altered gene will be sequenced in each case and then positioned in a plasmid so as to get active production of the enzyme by the bacterium. Papain will then be isolated and subsequently subjected to the QSAR analysis to determine what effect the amino acid substitution has had on the constants in the equations. All previous work on enzyme substrate binding has involved varying substituents on a substrate molecule to study enzyme/substrate interaction. This will represent a pioneering study in which the role of portions of the enzyme in substrate binding will be determined by varying the enzyme. At project's end, we hope to be making modifications in the enzyme that restrict the number of substrates accepted. The experimental system will enable refinement of the QSAR equations employed in drug design and will serve as a prototype for studies on the re-engineering of enzymes for maximum therapeutic benefit.