In lysozyme, the l&lactone form of sugar acids, having a half-chair conformation is thought to be an analog of the transition state in the enzyme-catalyzed reaction, and to be bound at Subsite D, which is situated at the catalytic sitele3 of the enzyme. Moreover, Hehre et aL4-* pointed out that D-glucal, an unusual saccharide, is a good analog of the intermediate, rather than being simply a substrate analog, in the enzyme catalyzed reaction of glycosidases. Thus, studies on the interaction of lactones with glucoamylase, one of the glycosidases, could be useful for investigation of the enzyme mechanism and characterization of the subsites of the enzyme.
D-Glucono-l$lactone (D-gluconolactone), which has a half-chair confor-mation6, is a potent inhibitor of glucoamylase, and is considered to be bound at Subsite 1, probably being mimic of the substrate conformation at the catalytic site during the transition state"'. On the other hand, when D-glucose and D-mannose were investigated for the inhibition of glucoamylase, it was concluded7*9 that these saccharides are bound at Subsite 2. Such binding is expected to result in purely competitive inhibition for substrates, including amylose'.
We now report studies on the interaction of glucoamylase with D-mannono-1,5-lactone (D-mannonolactone), which has a conformation similar to that of Dgluconolactone, and might be expected to be a potent inhibitor by binding at Subsite 1. However, from our results with inhibition steady-state kinetics, difference spectrophotometry, and the fluorescence method, we conclude that D-mannonolactone is not bound at Subsite 1, but may be bound at Subsite 2.