UV-induced vanadate-dependent modification and cleavage of skeletal myosin subfragment 1 heavy chain. 2. Oxidation of serine in the 23-kDa NH2-terminal tryptic peptide.
Myosin subfragment 1 (S1) can be specifically photomodified at the active site without polypeptide chain cleavage by irradiating the stable MgADP-orthovanadate-S1 complex with UV light above 300 nm [Grammer, J. C., Cremo, C. R., & Yount, R. G. (1988) Biochemistry (preceding paper in this issue)]. Here, the UV spectral properties of photomodified S1 were used to determine the nature and location of the photomodified residue(s) within S1. By comparison of the unusual pH dependence of the UV absorption spectrum of the photomodified S1 to that of the S1-MgADP-Vi complex as a control, the photomodified residue(s) was (were) localized to the 23-kDa NH2-terminal tryptic peptide of the heavy chain. NaBH4 reduced the photomodified S1, but not the control, to regenerate the original spectral properties and ATPase activities of the unmodified S1. Amino acid analysis of photomodified S1 reduced with NaB3H4 gave only [3H]serine, suggesting the hydroxyl group of serine had been oxidized to a "serine aldehyde". The pH dependence of the absorption spectrum of the photomodified enzyme can be explained by an equilibrium between a chromophoric enolate anion of the serine aldehyde (favored in base) and less chromophoric keto and enol forms (favored in acid). The oxidized serine(s) was (were) shown to be directly involved with the vanadate-dependent photocleavage of the S1 heavy chain previously described by Grammer et al. (1988). This serine(s) is (are) likely to be important to the binding and hydrolysis of the gamma-PO4 of ATP at the active site of S1.
Cremo
C R
CR
Institute of Biological Chemistry, Washington State University, Pullman 99164-4660.
Grammer
J C
JC
Yount
R G
RG
D000244:Adenosine Diphosphate; D000818:Animals; D001665:Binding Sites / Q000528:radiation effects; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D009132:Muscles / Q000032:analysis; D015879:Myosin Subfragments; D009218:Myosins / Q000528:radiation effects*; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000528:radiation effects*; D011817:Rabbits; D012694:Serine; D014357:Trypsin; D014466:Ultraviolet Rays; D014638:Vanadates
D015879:Myosin Subfragments; D010446:Peptide Fragments; D014638:Vanadates; D012694:Serine; D000244:Adenosine Diphosphate; D014357:Trypsin; D009218:Myosins