Determination of the Product Dissociation Constant of Adenosine Deaminase and Purification of Malate Synthase from Baker's Yeast
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The origin of the extraordinary catalytic efficiency of enzymes continues to be one of the major questions in biochemistry. Recent developments in spectroscopic methods (particularly Nuclear Magnetic Resonance (NMR) Spectroscopy and Fourier Transform Infrared (FTIR) Spectroscopy) make it possible to observe directly the intermediate structures (the transition state structures) which reflect the catalytic strategies used by an enzyme. The catalytic strategies employed by adenosine deaminase and malate synthase make them almost perfectly suited for investigation by these spectroscopic techniques. The quantitative analysis of C-13 NMR data of adenosine deaminase requires a very accurate knowledge of its product dissociation constant. Malate synthase, on the other hand, is not commercially available. This paper reports on full time-course, stopped-flow experiments designed to determine the product dissociation constant of adenosine deaminase and on purification of malate synthase from baker's yeast. By fitting simulated progress curves to experimentally obtained progress curves, the product dissociation constant of adenosine deaminase was determined to be 546.B±28.4 uM. Malate synthase from one-pound of baker's yeast was purified 250-fold to a specific activity of about 0.6 ukat/mg protein. Approximately 25 mg of purified protein was obtained within 10 days in a yield of 40% relative Lo the total activity in the cell-free crude extracts.