Alteration of the ATP hydrolysis and actin binding properties of thrombin-cut myosin subfragment 1.
We have characterized various structural and enzymatic properties of the (68K-30K)-S-1 derivative obtained by thrombic cleavage [Chaussepied, P., Mornet, D., Audemard, E., Derancourt, J., & Kassab, R. (1986) Biochemistry (preceding paper in this issue)]. The far-ultraviolet CD spectra and thiol reactivity measurements indicated an unchanged overall polypeptide conformation of the enzyme whereas the CD spectra in the near-ultraviolet region suggested a local change in the environments of phenylalanine side chains; the latter finding was rationalized by considering the existence of about five of these amino acids in the vicinity of the cleavage sites. When the binding of Mg2+-ATP and Mg2+-ADP to the derivative was assessed by CD spectroscopy, distinct spectra were obtained with the two nucleotides as with native subfragment 1 (S-1), but some spectral features were unique to the nicked S-1. Stern-Volmer fluorescence quenching studies using acrylamide and the analogues 1,N6-ethenoadenosine 5'-triphosphate and 1,N6-ethenoadenosine 5'-diphosphate indicated that the complexes formed with the modified S-1 have a solute quencher accessibility close to that observed for the complexes with the normal S-1. However, in contrast to the parent enzyme, the thrombin-cut S-1 was unable to bind irreversibly Mg2+-ATP, nor did it form a stable Mg2+-ADP-sodium vanadate complex or achieve the entrapping of Mg2+-ADP after cross-linking of SH1 and SH2 with N,N'-p-phenylenedimaleimide. Additionally, the amplitude of the Pi burst was very low, indicating that the inactivation of the proteolyzed S-1 was linked to the suppression of the hydrolysis step in the ATPase cycle.(ABSTRACT TRUNCATED AT 250 WORDS)
D000199:Actins / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002417:Cattle; D002918:Chymotrypsin / Q000378:metabolism; D002942:Circular Dichroism; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D007700:Kinetics; D008970:Molecular Weight; D009132:Muscles / Q000201:enzymology*; D015879:Myosin Subfragments; D009218:Myosins / Q000302:isolation & purification / Q000378:metabolism*; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D011817:Rabbits; D013917:Thrombin / Q000378:metabolism*
D000199:Actins; D015879:Myosin Subfragments; D010446:Peptide Fragments; D000255:Adenosine Triphosphate; D004228:Dithionitrobenzoic Acid; D002918:Chymotrypsin; D013917:Thrombin; D000251:Adenosine Triphosphatases; D009218:Myosins