While there is no question that ligands can induce large-scale domain movements that narrow (close) the active-site cleft of the catalytic (C) subunit of cAMP-dependent protein kinase (cAPK), the results from small-angle X-ray scattering, protein footprinting, and thermostability studies are inconsistent with regard to which ligands induce these movements. This inconsistency suggests a greater complexity of cAPK conformational dynamics than is generally recognized. As an initial step to study this issue in relation to the catalysis, a new method to measure cAPK domain closure was developed, and the state of domain closure and the local segmental flexibility at major steps of the cAPK catalytic cycle were examined with site-directed labeling and fluorescence spectroscopy. To achieve this, a C subunit mutant (F239C/C199A) was engineered that allowed for fluorescein 5-maleimide (donor) conjugation of F239C in the large lobe and tetramethylrhodamine (acceptor) conjugation of C343 in the small lobe. Domain closure was assessed as an increase in the efficiency of energy transfer between donor and acceptor. The anisotropy decay of fluoroscein 5-maleimide, conjugated to a site of cysteine substitution (K81C) in the small lobe of the C subunit was used to assess the local backbone flexibility around the B helix. The effects of substrate/pseudosubstrate (ATP and PKI(5-24)), a fragment of protein kinase inhibitor) and products (ADP and phosphorylated PKS) on domain closure and B helix flexibility were measured. The results show that domain closure is not tightly coupled to the flexibility around K81C. Moreover, although substrates/pseudosubstrate and products independently close the active-site cleft, only the substrates substantially decreased the backbone flexibility around the B helix. Because this order-to-disorder transition coincides with the phosphoryl transfer transition, the results suggest the existence of an internal entropy contribution to catalysis.
Copyright 2002 Academic Press.