An experiment on the chemical modification of essential tryptophan residues of barley β-amylase

Abstract

Introduction Enzymes bind their substrates through a specific limited area on the enzyme surface called the active site at which catalysis occurs. Apart from X-ray diffraction, the most successful approach to the study of the components and the threedimensional conformation of the active site is of modification of putative essential residues by group-specific chemical reagents, affinity labels etc. Small alterations in the structure may be detected from the reactivity of amino acid residues with specific reagents. Modification studies can give a better understanding of the catalytic mechanism or detect which amino acid residues are directly involved in the catalytic process. Kinetic studies together with chemical modification can give a more complete picture of the active site amino acids. As a complement to the study of enzyme catalysis and kinetics, UV-difference spectra can highlight the implication of certain essential residues in the active site. We have chosen barley 13-amylase (BA) whose activity can be measured very easily. I BA contains essential tryptophan residues which may be chemically modified by treatment with Nbromosuccinimide (NBS). These experiments described are used in a biochemistry practical session at the Department of Biochemistry, Gulbarga University at first-year MSc (Pre) level (intake 15 students a year). Another consideration is that the student should be able to complete the practical within 3-4 hours. The practical described here is a simple and straightforward demonstration of the various aspects of enzymes.