NMR-SPECTROSCOPY OF EXCHANGEABLE PROTONS OF GLUCOAMYLASE AND OF COMPLEXES WITH INHIBITORS IN THE 9-15-PPM RANGE

H-1-NMR spectra have been recorded for glucoamylases I and II from Asp ergillus awamori var. X100 and from A, niger in the 9-15-ppm region. A t least 17 distinct peaks, many of them arising from single protons, a re observed. These are designated A-Q, A being the furthest downfield. At least 9 of these are lost rapidly by exchange when the enzyme is p laced in D2O. Peaks A, B, E and H undergo distinct shifts with pH chan ge in the pH region 3-7. Several others undergo smaller shifts. Small differences are also seen between the enzymes from the two different s ources. Binding of the pseudotetrasaccharide inhibitor acarbose leads to a 0.50-ppm downfield shift of peak B, other smaller changes, and re tention of two additional protons in D2O. delta-D-Gluconolactone induc es shifts in peaks E, H, and L. The slow substrate maltitol causes pea k A to broaden and shift, peaks J and K to shift and a new or greatly shifted resonance to appear at 15.4 ppm. It disappears as the maltitol is hydrolyzed. Treatment with iodoacetamide of diethyl pyrocarbonate leads to disappearance of peak D at 12.3 ppm. When this peak was irrad iated strong nuclear Overhauser effects (NOE) were observed at 8.01 pp m and 7.22 ppm, positions expected for the C epsilon 1 and C delta 2 p rotons of an uncharged imidazole ring. We identify D as arising from t he N epsilon 2 proton of His254 which is uncharged except at the lowes t pH values. Other NOE and two-dimensional NOE spectra have provided a dditional information. Three mutant forms of the A. niger enzyme, in w hich tryptophan residues have been replaced by phenylalanine, have bee n examined. Because of shifts induced by changes in ring current and o ther environmental effects it is hard to make a direct identification of the resonances from the replaced indole NH protons. However, on the basis of a distinct NOE between peaks E and H we have identified thes e resonances as arising from the indole NH protons of Trp52 and Trp120 . Other possible assignments are considered. The NMR spectra of the gl ucoamylases I, which have a starch binding domain of about 104 residue s at the carboxyl terminus, show four sharp resonances in the 9.7-10.6 -ppm range that are not present in the glucoamylases II, which lack th is domain. These resonances no doubt represent the four indole NH ring protons from Trp543, Trp562, Trp590 and Trp615. Three of these are ve ry sharp suggesting a high mobility of this domain.