Interaction of a Spin-labeled Analogue of Nicotinamide Adenine Dinucleotide with Alcohol Dehydrogenase: III. Thermodynamic, Kinetic, and Structural Properties of Ternary Complexes as Determined by Nuclear Magnetic Resonance

Abstract

Abstract ADP-C5H4(CH3)4N(·)/—O (ADP-R·) is a paramagnetic analogue of NAD in which the unpaired electron is located in a region which corresponds to the pyridine N-ribose C1 bond of the coenzyme. Studies of the effect of ADP-R· on the longitudinal nuclear relaxation rate (1/T1) of the protons of have previously shown this analogue to form binary complexes with liver alcohol dehydrogenase and to form a ternary enzyme-ADP-R·-ethanol complex. By the same technique, ternary complexes are shown here with acetaldehyde and isobutyramide. The dissociation constants of all ternary complexes are in reasonable agreement with those reported for the respective kinetically active species. All of the ternary complexes are less effective in relaxing protons than is the binary e-ADP-R· complex, suggesting that ethanol, acetaldehyde, and isobutyramide displace molecules which are hydrogen-bonded to the paramagnetic nitroxide group of enzyme-bound ADP-R·. This is directly shown by the effect of e-ADP-R· on the transverse relaxation rates (1/T2) of the protons of ethanol, acetaldehyde, and isobutyramide. In the absence of enzyme, high concentrations of ADP-R· (5 mm) increase 1/T2 of the protons of ethanol. Its effect on the methylene protons is g3 times its effect on the methyl protons. The complex of ADP-R· with liver alcohol dehydrogenase at much lower concentrations (∼0.2 mm) relaxes the protons of ethanol and the effect on the methyl protons is 8 to 23 times greater than the effect on the methylene protons, suggesting that the enzyme reorients the ethanol with reference to the unpaired electron of ADP-R· in the ternary complex. Depending on conditions, the enzyme enhances the effect of ADP-R· on 1/T2 of the methylene protons by a factor of 1.5 to 13.4 and on the methyl protons by a factor of 16 to 148. The e-ADP-R· complex is comparably effective in relaxing the protons of acetaldehyde and of isobutyramide, indicating that the unpaired electron is very near the bound substrates and inhibitor in the respective ternary complexes. All of these effects are reversed by the addition of excess NADH. The temperature dependence of 1/T2 of the methyl protons of ethanol in the presence of the enzyme-ADP-R ·-ethanol complex can be fit by two rate processes: 1/τm, the rate of chemical exchange of ethanol molecules into the ternary complex, and 1/T2m, the relaxation rate of ethanol molecules which are in the ternary complex. From 1/τm, the exchange rate of ethanol into the ternary complex is 610 sec-1 at 25° which is two orders of magnitude faster than the maximum turnover number of the alcohol dehydrogenase reaction. Lower limits of 770 sec-1 and 180 sec-1 are set on the exchange rates of acetaldehyde and isobutyramide into their ternary complexes. From the dipolar contributions to 1/T2m, the average distances between the unpaired electron of enzyme-bound ADP-R · and the protons of the bound substrates and inhibitors, calculated from the Solomon-Bloembergen equation, are: ethanol (methyl), 3.6 A; ethanol (methylene), 4.1 A; acetaldehyde (aldehyde), 3.1 A; isobutyramide (methyl), ≤3.9 A. It is concluded that substrates bind to liver alcohol dehydrogenase on the water side of the bound coenzymes and directly overlie the ribosidic bond to pyridine. Structures consistent with the calculated distances permit molecular contact between the hydrogen-donating and hydrogen-accepting positions of the substrates and the coenzymes and, therefore, support the direct transfer of hydrogen between them. Although hydrogen transfer by way of stacked pyridine and indole rings can be excluded, though the participation of tryptophan in an edge to edge structure with the pyridine ring cannot be excluded.