Changes of Creatine Kinase Secondary Structure Induced by the Release of Nucleotides from Caged Compounds

Abstract

Light-induced release of ADP and ATP from their respective caged nucleotides produced small distinct difference infrared spectra of creatine kinase (CK), indicating that ADP and ATP binding to CK promoted different structural alteration. The positive band at 1638-1640 cm-' and the negative band at about 1650-1652 cm on the reaction-induced infrared difference spectra in the amide I region were insensitive to the deuteration effects. They were assigned to the peptide backbone of the ADPIATPbinding site. In addition P, or ATP binding produced another positive band at 1657-1659 cm-' corresponding to the C = O (amide I band) associated with the y-phosphate of ATP. This site was also affected when ADP was added, indicating coupling interactions between both sites. No additional structural changes were observed when creatine and ADP were added, suggesting that the creatine-binding site was uncoupled from the ADP-binding site. The infrared difference spectra of a transition-state-analog complex formed by the addition of ADP, creatine and NO; (a planar-phosphate-mimicking group) lacked the 1657 -1659-cm-' band indicating that the binding site of y-phosphate within CK, was not affected. Infrared changes in the 1560-1590-cm-' region suggested that carboxylate groups of Asp or Glu were involved in the binding of P,, ADP and ATP.

ADP‐Binding and ATP‐Binding Sites in Native and Proteinase‐K‐Digested Creative Kinase, Probed by Reaction‐Induced Difference Infrared Spectroscopy

“…Apart from minor changes in relative magnitude of the 'H/lH-exchange-sensitive band at 1555 cm-' (component of the amide-I1 band), which modified the baseline from 1500 cm-' to 1700 cm-', the main difference between the RIDS of native CK and that of nicked CK, is the shift of the broad band from 1666-1668 cm-' ( (Fig. 5 B ; Raimbault et al, 1996) or without the enzyme (Fig. 5 C ;Raimbault et al, 1996) were determined.…”

“…The three positive bands at 1637-1638, 1595-1597 and at 1573-1575 cm-', and the four negative bands at 1651-1652, 1625-1626, 1610-1612 and 1580-1583 cm-' resulted from the binding of ADP to CK and are related to the structural changes of the enzyme ( Fig. 3A; Raimbault et al, 1996). Addition of dithiothreitol (up to 10 mM) did not produce any shift of the protein bands but reduced their respective intensities for samples containing native- (Fig.…”

“…5 B ; Raimbault et al, 1996) or without the enzyme (Fig. 5 C ;Raimbault et al, 1996) were determined. Due to the strong water band around 1650 cm-I, the baseline in this region is not very flat (Fig.…”

Magnesium−Adenosine Diphosphate Binding Sites in Wild-type Creatine Kinase and in Mutants: Role of Aromatic Residues Probed by Raman and Infrared Spectroscopies

“…The 1637-1640 cm -1 band is attributed to a carbonyl group adjacent to residue that can interact with either the purine or ribose moieties of nucleotide. The 1648-1651, 1637-1640, and 1625-1626 cm -1 component bands in the RIDS of wildtype creatine kinase are almost insensitive to isotopic shift induced by changing from 1 H 2 O to 2 H 2 O buffers (46), suggesting that the carbonyl groups associated with these bands remained in the hydrophobic region. However, the 1612 cm -1 band (deuterated buffer) shifted to 1615 cm -1 (nondeuterated buffer), indicating an exposed carbonyl group (46).…”

“…The three positive bands located at 1666, 1657, and 1637-1640 cm -1 and the four negative bands located at 1648-1651, 1625, 1612-1615, and 1581-1584 cm -1 resulted from the effects of photorelease of Mg-ADP in the presence of mutant CK. On the basis of earlier works (22,33,46), the 1666-1668, 1657-1660, 1648-1651, 1625-1626, and 1612 cm -1 bands were assigned to carbonyl groups of peptide backbone adjacent to residues that can bind to the phosphate groups of the nucleotide moiety. The 1637-1640 cm -1 band is attributed to a carbonyl group adjacent to residue that can interact with either the purine or ribose moieties of nucleotide.…”