Tissue-nonspecific alkaline phosphatase is an anti-inflammatory nucleotidase.

Tissue-nonspecific alkaline phosphatase (TNAP) is necessary for skeletal mineralization by its ability to hydrolyze the mineralization inhibitor inorganic pyrophosphate (PPi), which is mainly generated from extracellular ATP by ectonucleotide pyrophosphatase phosphodiesterase 1 (NPP1). Since children with TNAP deficiency develop bone metaphyseal auto-inflammations in addition to rickets, we hypothesized that TNAP also exerts anti-inflammatory effects relying on the hydrolysis of pro-inflammatory adenosine nucleotides into the anti-inflammatory adenosine. We explored this hypothesis in bone metaphyses of 7-day-old Alpl+/- mice (encoding TNAP), in mineralizing hypertrophic chondrocytes and osteoblasts, and non-mineralizing mesenchymal stem cells (MSCs) and neutrophils, which express TNAP and are present, or can be recruited in the metaphysis. Bone metaphyses of 7-day-old Alpl+/- mice had significantly increased levels of Il-1ß and Il-6 and decreased levels of the anti-inflammatory Il-10 cytokine as compared with Alpl+/+ mice. In bone metaphyses, murine hypertrophic chondrocytes and osteoblasts, Alpl mRNA levels were much higher than those of the adenosine nucleotidases Npp1, Cd39 and Cd73. In hypertrophic chondrocytes, inhibition of TNAP with 25 µM of MLS-0038949 decreased the hydrolysis of AMP and ATP. However, TNAP inhibition did not significantly modulate ATP- and adenosine-associated effects in these cells. We observed that part of TNAP proteins in hypertrophic chondrocytes was sent from the cell membrane to matrix vesicles, which may explain why TNAP participated in the hydrolysis of ATP but did not significantly modulate its autocrine pro-inflammatory effects. In MSCs, TNAP did not participate in ATP hydrolysis nor in secretion of inflammatory mediators. In contrast, in neutrophils, TNAP inhibition with MLS-0038949 significantly exacerbated ATP-associated activation and secretion of IL-1ß, and extended cell survival. Collectively, these results demonstrate that TNAP is a nucleotidase in both hypertrophic chondrocytes and neutrophils, and that this nucleotidase function is associated with autocrine effects on inflammation only in neutrophils.

Autocrine stimulation of osteoblast activity by Wnt5a in response to TNF-α in human mesenchymal stem cells.

Although anti-tumor necrosis factor (TNF)-α treatments efficiently block inflammation in ankylosing spondylitis (AS), they are inefficient to prevent excessive bone formation. In AS, ossification seems more prone to develop in sites where inflammation has resolved following anti-TNF therapy, suggesting that TNF-α indirectly stimulates ossification. In this context, our objectives were to determine and compare the involvement of Wnt proteins, which are potent growth factors of bone formation, in the effects of TNF-α on osteoblast function. In human mesenchymal stem cells (MSCs), TNF-α significantly increased the levels of Wnt10b and Wnt5a. Associated with this effect, TNF-α stimulated tissue-non specific alkaline phosphatase (TNAP) and mineralization. This effect was mimicked by activation of the canonical ß-catenin pathway with either anti-Dkk1 antibodies, lithium chloride (LiCl) or SB216763. TNF-α reduced, and activation of ß-catenin had little effect on expression of osteocalcin, a late marker of osteoblast differentiation. Surprisingly, TNF-α failed to stabilize ß-catenin and Dkk1 did not inhibit TNF-α effects. In fact, Dkk1 expression was also enhanced in response to TNF-α, perhaps explaining why canonical signaling by Wnt10b was not activated by TNF-α. However, we found that Wnt5a also stimulated TNAP in MSCs cultured in osteogenic conditions, and increased the levels of inflammatory markers such as COX-2. Interestingly, treatment with anti-Wnt5a antibodies reduced endogenous TNAP expression and activity. Collectively, these data suggest that increased levels of Dkk1 may blunt the autocrine effects of Wnt10b, but not that of Wnt5a, acting through non-canonical signaling. Thus, Wnt5a may be potentially involved in the effects of inflammation on bone formation.

Ankylosing spondylitis, late osteoarthritis, vascular calcification, chondrocalcinosis and pseudo gout: toward a possible drug therapy.

In this review we consider diseases associated with pathological mineralization/ossification, namely, ankylosing spondylitis (AS), osteoarthritis (OA), generalized artery calcification of infancy (GACI), vascular calcification as well as chondrocalcinosis (CC) and pseudo gout. Deciphering the key enzymes implicated in the calcification process is an objective of prime importance and the ultimate goal is to synthesize inhibitors of these enzymes in order to provide efficient alternate therapeutic strategies that will slow down the pathologic mineralization and complement the arsenal of anti-inflammatory drugs. One of the difficulties in the definition of diseases associated with pathologic mineralization/ossification lies in the controversial relationship between the type of calcification and the nature of the disease. Here, we propose to clarify this relationship by making a distinction between diseases associated with hydroxyapatite (HA) and calcium pyrophosphate dihydrate (CPPD) deposits. AS, OA, GACI and vascular calcification are usually characterized by mineralization/ossification associated with HA deposits, while CC and pseudo gout are mostly characterized by CPPD deposits. Although both HA and CPPD deposits may occur concomitantly, as in chronic pyrophosphate arthritis or in OA with CPPD, they are formed as a result of two antagonistic processes indicating that treatment of distinct diseases can be only achieved by disease-specific drug therapies. The hydrolysis of PPi, an inhibitor of HA formation, is mostly controlled by tissue non-specific alkaline phosphatase TNAP, while PPi production in the extracellular medium is controlled by ANK, a PPi transporter, and/or NPP1 which generates PPi from nucleotide triphosphates. Low PPi concentration may lead to a preferential deposition of HA while high PPi concentration will favor the formation of CPPD deposits. Thus, HA and CCPD deposition cannot occur concomitantly because they are determined by the Pi/PPi ratio which, in turn, depends on the relative activities of antagonistic enzymes, TNAP hydrolyzing PPi or ANK and NPP1 producing PPi. TNAP inhibitors could prevent HA formation in AS, in late OA, in GACI, as well as in vascular calcifications, while ANK or NPP1 inhibitors could slow down CCPD deposition in CC and pseudo gout.

Inorganic pyrophosphate as a regulator of hydroxyapatite or calcium pyrophosphate dihydrate mineral deposition by matrix vesicles.

OBJECTIVE: Pathological mineralization is induced by unbalance between pro- and anti-mineralization factors. In calcifying osteoarthritic joints, articular chondrocytes undergo terminal differentiation similar to that in growth plate cartilage and release matrix vesicles (MVs) responsible for hydroxyapatite (HA) or calcium pyrophosphate dihydrate (CPPD) deposition. Inorganic pyrophosphate (PP(i)) is a likely source of inorganic phosphate (P(i)) to sustain HA formation when hydrolyzed but also a potent inhibitor preventing apatite mineral deposition and growth. Moreover, an excess of PP(i) can lead to CPPD formation, a marker of pathological calcification in osteoarthritic joints. It was suggested that the P(i)/PP(i) ratio during biomineralization is a turning point between physiological and pathological mineralization. The aim of this work was to determine the conditions favoring either HA or CPPD formation initiated by MVs. METHODS: MVs were isolated from 17-day-old chicken embryo growth plate cartilages and subjected to mineralization in the presence of various P(i)/PP(i) ratios. The mineralization kinetics and the chemical composition of minerals were determined, respectively, by light scattering and infrared spectroscopy. RESULTS: The formation of HA is optimal when the P(i)/PP(i) molar ratio is above 140, but is completely inhibited when the ratio decreases below 70. The retardation of any mineral formation is maximal at P(i)/PP(i) ratio around 30. CPPD is exclusively produced by MVs when the ratio is below 6, but it is inhibited for the ratio exceeding 25. CONCLUSIONS: Our findings are consistent with the P(i)/PP(i) ratio being a determinant factor leading to pathological mineralization or its inhibition.

A comparative analysis of strategies for isolation of matrix vesicles.

Matrix vesicles (MVs) are extracellular organelles involved in the initial steps of mineralization. MVs are isolated by two methods. The first isolation method of MVs starts with collagenase digestion of osseous tissues, followed by two differential centrifugations. The second isolation method does not use proteases but rather starts with differential centrifugation, followed by a fractionation on a sucrose gradient. The first method results in a homogeneous population of MVs with higher cholesterol/lipid content, alkaline phosphatase activity, and mineral formation rate as compared with MVs isolated by the second method. The second method leads to higher protein diversity as compared with MVs isolated according to the first method. Due to their distinct protein composition, lipid-to-protein and cholesterol-to-phospholipid ratios, and differences in rates of mineral formation, both types of isolated MVs are crucial for proteomic analysis and for understanding the regulation of mineralization process at the molecular level.

Monitoring of secondary and tertiary structure changes in the gastric H+/K+-ATPase by infrared spectroscopy.

Conformational changes occurring in the catalytic cycle of the H+/K+-ATPase were monitored by Fourier transform infrared spectroscopy (FTIR). Caged compounds were used to release ATP, in the presence of Ca2+, to induce the transition between the E1 and E1-P conformation of the H+/K+-ATPase. In addition to bands associated with the photolysis of the caged compounds, some peaks of the difference infrared spectra were associated with changes in secondary structure and modifications of the ionization in the side chains of amino-acid residues (Glu or Asp). These changes were specific to the reaction between the ligand and the enzyme. We estimated that 39 amino acids changed their secondary structure during the reaction and four amino-acid residues were deprotonated. Similar spectral changes appeared when ADP was released from its precursor. The release of Pi from the same caged molecule did not induce similar changes. Changes in tertiary structure occurring during the binding of adenosine and phosphorylation of the enzyme were demonstrated by recording hydrogen/deuterium exchange kinetics by attenuated total reflectance FTIR spectroscopy (ATR-FTIR). At least 129 amide protons were involved in a tertiary structure change induced by ATP. This suggested that secondary structure change transduced a much larger tertiary structure modification.

Mitochondrial creatine kinase binding to phospholipids decreases fluidity of membranes and promotes new lipid-induced beta structures as monitored by red edge excitation shift, laurdan fluorescence, and FTIR.

Structural modifications induced by the binding of mitochondrial creatine kinase (mtCK) to saturated and unsaturated phospholipids were monitored by using Laurdan, a membrane probe sensitive to the polarity of the environment. The abrupt change characteristic of a phase transition of lipids alone was attenuated by addition of mtCK. Generalized polarization spectra indicated that mtCK surface binding changed the phospholipid liquid-crystalline state to a more rigid state. Infrared spectra of lipids further strengthened these results: upon mtCK binding, the phospholipid methylene chains had a more rigid conformation than that observed without mtCK at the same temperature. After mtCK binding to vesicles of perdeuterated dimyristoylphosphatidylcholine and nondeuterated dimyristoylphosphatidylglycerol, no lateral phase separation was observed, suggesting that both lipids were rigidified. Moreover, mtCK bound to liposomes exhibited an uncommon red edge excitation shift of 19 nm, while that of the soluble enzyme was only 6 nm. These results indicated that the environment of some mtCK tryptophan residues was motionally restricted. Strong stabilization of the enzyme structure against heat denaturation was observed upon lipid binding. In addition, lipids promoted a new reversible protein-protein or protein-lipid interaction, as evidenced by infrared data showing a slight modification of the beta sheet over alpha helix ratio with formation of a new 1632-cm(-)(1) beta sheet instead of the soluble protein 1636-cm(-)(1) one. Such modifications, inducing a decrease in the fluidity of the mitochondrial membranes, may play a role in vesicle aggregation; they could be implicated in the appearance of contact sites between internal and external mitochondrial membranes.

Structural changes of mitochondrial creatine kinase upon binding of ADP, ATP, or Pi, observed by reaction-induced infrared difference spectra.

Structural modifications of rabbit heart mitochondrial creatine kinase induced by the binding of its nucleotide substrates and Pi were investigated. Reaction-induced difference spectra (RIDS), resulting from the difference between infrared spectra recorded before and after the photorelease of a caged ligand, allow us to detect very small variations in protein structure. Our results indicated that the protein secondary structure remained relatively stable during nucleotide binding. Indeed, this binding to creatine kinase affected only a few amino acids, and caused small peptide backbone deformations and alterations of the carbonyl side chains of aspartate or glutamate, reflecting modifications within preexisting elements rather than a net change in secondary structure. Nonetheless, MgADP and MgATP RIDS were distinct, whereas the MgPi RIDS presented some similarities with the MgATP one. The difference between MgADP and MgATP RIDS could reflect a distinct configuration of the two metal-nucleotide complexes inducing a different positioning and/or a distinct binding mode to the creatine kinase active site. Comparison of the MgATP and MgPi RIDS suggests that Pi binding took place at the same binding site as the gamma-phosphoryl group of ATP. Thus, the difference between MgADP and MgATP RIDS would mainly be due to the effect of the gamma-P of ATP. The differences observed when comparing the RIDS resulting from the binding of nucleotides to octameric mitochondrial creatine kinase or dimeric cytosolic isoform could reflect the distinct oligomerization states and physicochemical or kinetic properties of the two isoenzymes.

Annexins as nucleotide-binding proteins: facts and speculations.

Annexins are ubiquitous multifunctional Ca2+ and phospholipid-binding proteins whose mechanism of function remains largely unknown. The accumulated in vitro experimental evidence indicates that ATP and GTP are functional ligands for nucleotide-sensitive annexin isoforms. Such nucleotide binding could modulate Ca2+ homeostasis, vesicular transport and/or signal transduction pathways and link them to cellular energy metabolism. Alternatively, since annexins are able to interact with other nucleotide-utilizing proteins, such as various kinases, GTPases and structural proteins, these proteins could influence the guanine nucleotide exchange metabolism and/or control the activity of various G proteins. The nucleotide-binding properties of annexins may affect the development or maintenance of some pathologies and diseases in which changes in physiological concentrations of purine nucleotides or disruption of Ca2+ homeostasis are crucial targets.

Mitochondrial creatine kinase binding to liposomes and vesicle aggregation: effect of cleavage by proteinase K.

Mitochondrial creatine kinase and its proteinase K nicked-derivative interaction with liposomes induced slight secondary structure changes evidenced by infrared spectra. In nondenaturing conditions, the N-terminal (K1) and the C-terminal (K2) fragments remained associated with each other and bound to liposomes. When the two fragments were separated by denaturation, K2 was soluble, whereas most of K1 was adsorbed onto liposomes. The three-dimensional structure of uncleaved mtCK suggests that the C-terminal moiety, which contains positively charged surface residues, interacted with membranes. After denaturation and renaturation of the nicked enzyme, both peptides did not refold properly and did not reassociate with each other. The misfolded K1 fragment bound to the membrane through a stretch of positive residues, which were buried in the native enzyme. The lack of binding of the ill-folded K2 peptide could be related to the disruption of the optimal disposition of its positive charges, responsible for the correct interaction of native mtCK with membrane.

Magnesium-adenosine diphosphate binding sites in wild-type creatine kinase and in mutants: role of aromatic residues probed by Raman and infrared spectroscopies.

Two distinct methods were used to investigate the role of Trp residues during Mg-ADP binding to cytosolic creatine kinase (CK) from rabbit muscle: (1) Raman spectroscopy, which is very sensitive to the environment of aromatic side-chain residues, and (2) reaction-induced infrared difference spectroscopy (RIDS) and photolabile substrate (ADP[Et(PhNO(2))]), combined with site-directed mutagenesis on the four Trp residues of CK. Our Raman results indicated that the environment of Trp and of Tyr were not affected during Mg-ADP binding to CK. Analysis of RIDS of wild-type CK, inactive W227Y, and active W210,217,272Y mutants suggested that Trp227 was not involved in the stacking interactions. Results are consistent with Trp227 being essential to prevent water molecules from entering in the active site [as suggested by Gross, M., Furter-Graves, E. M., Wallimann, T., Eppenberger, H. M., and Furter, R. (1994) Protein Sci. 3, 1058-1068] and that another Trp could in addition help to steer the nucleotide in the binding site, although it is not essential for the activity of CK. Raman and infrared spectra indicated that Mg-ADP binding does not involve large secondary structure changes. Only 3-4 residues absorbing in the amide I region are directly implicated in the Mg-ADP binding (corresponding to secondary structure changes less than 1%), suggesting that movement of protein domains due to Mg-nucleotide binding do not promote large secondary structure changes.

Alzheimer's disease: its origin at the membrane, evidence and questions.

Numerous results on membrane lipid composition from different regions of autopsied Alzheimer's disease brains in comparison with corresponding fractions isolated from control brains revealed significant differences in serine- and ethanolamine-containing glycerophospholipid as well as in glycosphingolipid content. Changes in membrane lipid composition are frequently accompanied by alterations in membrane fluidity, hydrophobic mismatch, lipid signaling pathways, transient formation and disappearance of lipid microdomains, changes in membrane permeability to cations and variations of other membrane properties. In this review we focus on possible implications of altered membrane composition on beta-amyloid precursor protein (APP) and on proteolysis of APP leading eventually to the formation of neurotoxic beta-amyloid (A beta) peptides, the major proteinaceous component of extracellular senile plaques, directly involved in Alzheimer's disease pathogenesis.

ATP-Binding site of annexin VI characterized by photochemical release of nucleotide and infrared difference spectroscopy.

Structural changes induced by nucleotide binding to porcine liver annexin VI (AnxVI) were probed by reaction-induced difference spectroscopy (RIDS). Photorelease of the nucleotide from ATP[Et(PhNO2)] produced RIDS of AnxVI characterized by reproducible changes in the amide I region. The magnitude of the infrared change was comparable to RIDS of other ATP-binding proteins, such as Ca(2+)-ATPase and creatine and arginine kinases. Analysis of RIDS revealed the existence of ATP-binding site(s) (K(d) < 1 microM) within the AnxVI molecule, comprising five to six amino acid residues located in the C-terminal portion of the protein molecule. The binding stoichiometry of ATP:AnxVI was determined as 1:1 (mol/mol). ATP, in the presence of Ca2+, induced changes in protein secondary structure reflected by a 5% decrease in alpha-helix content of the protein in favor of unordered structure. Such changes may influence the affinity of AnxVI for Ca2+ and modulate its interaction with membranes.

Annexin VI: an intracellular target for ATP.

Annexin VI (AnxVI), an Ca2+- and phospholipid-binding protein, interacts in vitro with ATP in a calcium-dependent manner. Experimental evidence indicates that its nucleotide-binding domain which is localized in the C-terminal half of the protein differs structurally from ATP/GTP-binding motifs found in other nucleotide-binding proteins. The amino-acid residues of AnxVI directly involved in ATP binding have not been yet defined. Binding of ATP to AnxVI induces changes in the secondary and tertiary structures of protein, affecting the affinity of AnxVI for Ca2+ and, in consequence, influencing the Ca2+-dependent activities of AnxVI: binding to F-actin and to membranous phospholipids, and self-association of the annexin molecules. These observations suggest that ATP is a functional ligand for AnxVI in vivo, and ATP-sensitive AnxVI may play the role of a factor coupling vesicular transport and calcium homeostasis to cellular metabolism.

ADP-binding and ATP-binding sites in native and proteinase-K-digested creatine kinase, probed by reaction-induced difference infrared spectroscopy.

Conformational changes induced by nucleotide binding to native creatine kinase (CK) from rabbit muscle and to proteinase-K-digested (nicked) CK, were investigated by infrared spectroscopy. Photochemical release of ATP from ATP[Et(PhNO2)] in the presence of creatine and native CK produced reaction-induced difference infrared spectra (RIDS) of CK related to structural changes of the enzyme that paralleled the reversible phosphoryl transfer from ATP to creatine. Similarly the photochemical release of ADP from ADP[Et(PhNO2)] in the presence of phosphocreatine and native CK allowed us to follow the backward reaction and its corresponding RIDS. Infrared spectra of native CK indicated that carboxylate groups of Asp or Glu, and some carbonyl groups of the peptide backbone are involved in the enzymatic reaction. Native and proteinase nicked CK have similar Stokes' radii, tryptophan fluorescence, fluorescence fraction accessible to iodide, and far-ultraviolet CD spectra, indicating that native and modified enzymes have the same quaternary structures. However, infrared data showed that the binding site of the gamma-phosphate group of the nucleotide was affected in nicked CK compared with that of the native CK. Furthermore, the infrared absorptions associated with ionized carboxylate groups of Asp or Glu amino acid residues were different in nicked CK and in native CK.

Conformational changes of arginine kinase induced by photochemical release of nucleotides from caged nucleotides--an infrared difference-spectroscopy investigation.

The conformations of arginine kinase (AK) in AK x Mg x ADP, AK x Mg x ATP, AK x Mg x ADP x NO3-, AK x Mg x ADP x Arg and AK x Mg x ADP x NO3- x Arg complexes were investigated by measuring their reaction-induced infrared difference spectra (RIDS). The photochemical release of ATP from ATP[Et(PhNO2)] and of ADP from ADP[Et(PhNO2)] produced distinct RIDS of AK complexes, suggesting that binding of ADP and ATP promoted different structural alterations of the enzyme active-site. Small infrared changes in the amide-I region were observed, indicating that about 5-10 amino acid residues were involved in the nucleotide-binding site. These infrared changes were due to the structural alteration of the peptide backbone caused by the nucleotide-binding and to the coupling effects between the nucleotide-binding site and the other substrate (Arg or NO3-)-binding site. ATP binding to AK (as well as ADP-binding to AK in the presence of NO3-) induced protonation of a carboxylate group of Asp or Glu, as evidenced by the appearance of the 1733-cm(-1) band, which was not observed with the AK x Mg x ADP, AK x Mg x ADP x Arg and AK x Mg x ADP x NO3- x Arg complexes. The RIDS of the AK x Mg x ADP x NO3- x Arg complex showed new infrared bands at 1622 cm(-1) (negative) and at 1613 cm(-1) (positive), which were not seen in the RIDS of other complexes (without NO3- or/and Arg). In the transition-state-analog complex of AK, no protonation of the carboxylate residue (Asp or Glu) was observed, and the binding site of NO3- or the gamma-phosphate group of nucleotide was altered.

Purification and spectroscopic characterization of beta-amyloid precursor protein from porcine brains.

Soluble and membrane-bound isoforms of beta-amyloid protein precursor (APP) of Alzheimer's disease were extracted and purified from porcine brains. At least three types of soluble APP and membrane-bound APP with different molecular masses, ranging from 86 kDa to 116 kDa, were obtained. CD and infrared spectroscopies were used to determine the overall secondary-structure content of APP. The infrared spectra of soluble and membrane-bound APP (in dry and hydrated states) were similar in the amide-I and amide-II regions, suggesting that the overall secondary structures of the soluble and membrane isoforms were roughly identical. The amide-I band is composed of at least five component bands, located at 1694, 1674, 1652, 1637 and 1618 cm(-1) for soluble APP, and located at 1687, 1674, 1651, 1637 and 1614-1606 cm(-1) for membrane-bound APP, as evidenced by their respective second-derivative infrared spectra. The 1651-1652-cm(-1) band was associated with alpha-helix structures, while two types of beta-sheet structures are evidenced by two characteristic pairs of component bands. The 1674-cm(-1) and 1637-cm(-1) bands for soluble APP and membrane-bound APP were tentatively associated to beta-sheet structures. The second pair of bands, located at 1694 cm(-1) and at 1618 cm(-1) for soluble APP and at 1687 cm(-1) and 1614-1606 cm(-1) for membrane-bound APP, were associated with intermolecular beta-sheet structures or aggregated strands, as confirmed by heat denaturation. CD spectra indicated the presence of alpha-helix structures in soluble and membrane-bound APP. The secondary-structure content, estimated from CD spectra, was about 40-45% alpha-helix and 15-20% beta-sheet structures for soluble and membrane-bound APP.

Nucleotide binding sites in wild-type creatine kinase and in W227Y mutant probed by photochemical release of nucleotides and infrared difference spectroscopy.

Structural changes induced by nucleotide binding to the wild-type rabbit muscle creatine kinase (CK) and to its W227Y mutant were compared and probed by reaction-induced difference spectroscopy (RIDS). The reaction was induced by the photorelease of nucleotide from the caged nucleotides ADP[Et(PhNO2)] or ATP[Et(PhNO2)], producing the RIDS of CK. The concomitant addition of a saturated concentration of nucleotide and caged nucleotide modified the RIDS of CK, permitting structural changes caused by nucleotide binding in the wild-type creatine kinase to be identified. The W227Y mutant was inactive and its nucleotide binding site was partially impaired as shown by the disappearance or decrease of several nucleotide-sensitive bands in the RIDS of W227Y mutant. The magnitude of the decrease was not the same for each band, suggesting that distinct groups of W227Y mutant were affected differently during nucleotide binding. More precisely, the binding sites for gamma-phosphate and beta-phosphate of the nucleotide were not accessible in W227Y mutant as shown by the absence of the phosphate-sensitive 1666-1667-cm(-1) and 1625-cm(-1) bands in the RIDS of W227Y mutant. However the binding site of other parts of the nucleotide was partially accessible, since the 1638-1639-cm(-1) phosphate-insensitive band did not completely vanish in the RIDS of W227Y mutant. The RIDS of W227Y mutant with ADP[Et(PhNO2)] and creatine lacked the 1613-cm(-1) and 1581-cm(-1) bands, associated with vibrational modes of creatine, suggesting that coupling between the binding sites of the nucleotide and of creatine was altered in W227Y mutant. These results are in accordance with the earlier suggestions that residue W227 in CK is essential for preventing water molecules from penetrating into the active site and for orienting nucleotide in the binding site, by forming stacking interactions between its indole group and purine of the nucleotide and its indole group.

Changes of creatine kinase secondary structure induced by the release of nucleotides from caged compounds. An infrared difference-spectroscopy study.

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-1 and the negative band at about 1650-1652 cm-1 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 ADP/ATP-binding site. In addition Pi or ATP binding produced another positive band at 1657-1659 cm-1 corresponding to the C = O (amide I band) associated with the gamma-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 NO3- (a planar-phosphate-mimicking group) lacked the 1657-1659-cm-1 band indicating that the binding site of gamma-phosphate within CK, was not affected. Infrared changes in the 1560-1590-cm-1 region suggested that carboxylate groups of Asp or Glu were involved in the binding of Pi, ADP and ATP.