Tryptic proteol ysis of coupling factor-latent ATPase from Mycobacterium phlei. Theoretical modeling of structure-function relationships

Trypsin treatment of solubilized coupling factor-latent ATPase from Mycobacterium phlei alters its subunit structure and functional properties. This coupling factor exhibits ATPase activity following trypsin treatment. Concurrently, both the ability of the enzyme to rebind to membranes depleted of coupling factor and its capacity for coupled phosphorylation are lost. The native alpha (64 000 dalton) subunit undergoes limited proteolytic digestion, and the delta (14 000 dalton) subunit is partially lost. During the course of tryptic proteolysis, the coupling factor molecule may exist in one of ten unique structural states (e.g. the native, ATPase-inactive molecule exists in the ααα state). Rigorous analysis of the experimental data by theoretical modeling provided information concerning the intermediate structural states leading to the fully ATPase-activated α″α″α″ state under different conditions of trypsin treatment. The theoretical models of structure-function relationships that best-represented the experimental data predicted that the native coupling factor molecule contains three copies of the α (64 000 dalton) form of the alpha subunit, that the α″ (58 000 dalton) alpha subunit species contributes maximally and the α′ (61 000 dalton) form about half-maximally to ATPase activity, that membrane rebinding ability is proportional to the number of native alpha subunits in the enzyme, and that at least one native α subunit/molecule is required for full expression of coupled phosphorylation. These results indicate an essential role for the alpha subunit in the regulation of ATPase activity and in the ability of the solubilized coupling factor to rebind to depleted membranes.