Determinants of cysteine pK<sub>a</sub> values in creatine kinase and α1‐antitrypsin

Naor, Mor M.; Jensen, Jan H.

doi:10.1002/prot.20261

Cited by 34 publications

(42 citation statements)

References 25 publications

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“…The factors contributing to the low pK a value are not all immediately apparent, although it was shown that a S…OH bond between Cys283 and Ser285 ( Figure 2A) contributed about 1 pH unit to the lowered pK a value (19). This experimental result was in good agreement with the calculations of Naor and Jensen (25) who also hypothesized that the hydrogen bond between Cys283 and the amide of Ser285 would contribute approximately 1.5 pH units. Removal of these two hydrogen bonds would be expected to increase the pK a value of Cys283 to ~8.1, which is still below the standard pK a value for a cysteine residue.…”

supporting

confidence: 86%

“…Since the experimental geometries of the P284A and C283S/S285C mutants were not available, the structural models of the mutants were constructed from the wild type model (with Cys deprotonated) used by Naor and Jensen (25) (Figure 2B). In the case of the C283S/S285C variant the S and O atoms were interchanged ( Figure 2C).…”

Section: Protein Model Constructionmentioning

confidence: 99%

“…For the P284A mutant the methylene units in the proline ring were replaced by a single methyl group ( Figure 2D), followed by energy minimization of all atoms except the alpha carbons of Thr282 and Ser285. The model uses the coordinates from PDB 1QK1 for mitochondrial (30) creatine kinase as the relatively low resolution (3.5 Å) structure of HMCK had been shown previously to give erroneous results (25).…”

Section: Protein Model Constructionmentioning

confidence: 99%

“…In the acid (protonated) models, four positions of the cysteine proton (added manually) and the serine hydroxyl hydrogen atom were considered (25). The total free energy of the acid form was taken to be the "conformational average" of the free energies of each conformer (G i ), eq 1, (31): (1) where G 0 is the lowest energy conformer, and ΔG i = G i -G 0 .…”

Section: Protein Model Constructionmentioning

confidence: 99%

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Exploring the Role of the Active Site Cysteine in Human Muscle Creatine Kinase

et al. 2006

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All known guanidino kinases contain a conserved cysteine residue that interacts with the nonnucleophilic η 1 -nitrogen of the guanidino substrate. Site-directed mutagenesis studies have shown that this cysteine is important, but not essential for activity. In human muscle creatine kinase (HMCK) this residue, Cys283, forms part of a conserved cysteine-proline-serine (CPS) motif and has a pK a about 3 pH units below that of a regular cysteine residue. Here we employ a computational approach to predict the contribution of residues in this motif to the unusually low cysteine pK a . We calculate that hydrogen bonds to the hydroxyl and to the backbone amide of Ser285 would both contribute ~1 pH unit, while the presence of Pro284 in the motif lowers the pK a of Cys283 by a further 1.2 pH units. Using UV difference spectroscopy the pK a of the active site cysteine in WT HMCK and in the P284A, S285A and C283S/S285C mutants was determined experimentally. The pK a values, although consistently about 0.5 pH units lower, were in broad agreement with those predicted. The effect of each of these mutations on the pH-rate profile was also examined. The results show conclusively that, contrary to a previous report Biochemistry 40, 11698-11705), Cys283 is NOT responsible for the pK a of 5.4 observed in the WT V/K creatine pH profile. Finally we use molecular dynamics simulations to demonstrate that, in order to maintain the linear alignment necessary for associative inline transfer of a phosphoryl group, Cys283 needs to be ionized.Creatine kinase (CK, E.C. 2.7.3.2) catalyzes the reversible transfer of the γ-phosphoryl group of ATP to creatine (Cr), forming ADP and phosphocreatine (PCr). The latter is considered to be a reservoir of "high-energy phosphate" which is able to supply ATP, the primary energy source in bioenergetics, on demand. As a result, CK plays a major role in energy homeostasis of cells with intermittently high energy requirements, such as skeletal and cardiac muscle, neurons, photoreceptors, spermatozoa and electrocytes (1-3). CK is found in all vertebrates and exists in a variety of isoforms including the muscle, brain and mitochondrial isozymes. The two mitochondrial isozymes, ubiquitous (Mi u ) and sarcomeric (Mi s ), can exist as dimers but are generally octameric. The subunits of the muscle (M) and brain (B) isozymes, each with a molecular mass of ~43 kDa, form homodimers (MM, BB). In addition, they form a heterodimer (MB) which is used as a marker for myocardial infarction (4,5). In fact, cellular CK levels are perturbed in a number of human disease states including neurodegenerative diseases (6,7), muscular dystrophies (8) and cancer (9-11).

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supporting

confidence: 86%

Section: Protein Model Constructionmentioning

confidence: 99%

Section: Protein Model Constructionmentioning

confidence: 99%

Section: Protein Model Constructionmentioning

confidence: 99%

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Exploring the Role of the Active Site Cysteine in Human Muscle Creatine Kinase

et al. 2006

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“…Hydrogen bonds between amide nitrogen atoms and thiolate anions remain strong with interatomic distances of 3.4-4.0 Å. 25 Similar distances are routinely observed between the glutathione and other HPGDS inhibitors in this series. The physical properties of the compound were evaluated, and it was found that 8 had a solubility of 1.5 μg/mL (3.9 μM) at pH 6.50.…”

supporting

confidence: 75%

Discovery of an Oral Potent Selective Inhibitor of Hematopoietic Prostaglandin D Synthase (HPGDS)

Carron

Trujillo

et al. 2010

ACS Med. Chem. Lett.

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Hematopoietic prostaglandin D synthase (HPGDS) is primarly expressed in mast cells, antigen-presenting cells, and Th-2 cells. HPGDS converts PGH 2 into PGD 2 , a mediator thought to play a pivotal role in airway allergy and inflammatory processes. In this letter, we report the discovery of an orally potent and selective inhibitor of HPGDS that reduces the antigen-induced response in allergic sheep.KEYWORDS Hematopoietic prostaglandin D synthase (HPGDS), PGH 2 , PGD 2 , airway allergy, inflammatory processes, cyclooxygenase (COX) A sthma is a chronic inflammatory disorder of the airways that causes recurrent episodes of wheezing, breathlessness, chest tightness, and coughing in susceptible individuals. 1,2 Prostaglandin D 2 (PGD 2 ), a mediator of allergy and inflammation, is produced by mast cells and Th-2 cells in a variety of human tissues. PGD 2 is the most abundant de novo cyclooxygenase-derived mediator produced following IgE-mediated degranulation of mast cells. 3 The mast cell, following allergen-provoked degranulation, is believed to be the major source of PGD 2 found in the nasal exudates of patients with allergic rhinitis. PGD 2 levels increase dramatically in bronchoalveolar lavage fluid following allergen challenge, and the observation that patients with asthma exhibit bronchoconstriction upon inhalation of PGD 2 underscores the pathologic consequences of high levels of PGD 2 in the lung. 4 Treatment with PGD 2 produces significant nasal congestion and fluid secretion in man and dogs, and PGD 2 is 10 times more potent than histamine and 100 times more potent than bradykinin in producing nasal blockage in humans, demonstrating a role for PGD 2 in allergic rhinitis. 5,6 Mast cell-derived PGD 2 exerts its effect by activating two distinct G-protein-coupled receptors (GPCRs): the DP-1 receptor, a member of the prostanoid receptor subfamily, and the recently discovered chemoattractant receptor-homologous molecule expressed on T-helper 2 cells (CRTH 2 receptor). The DP-1 receptor is localized on the nasal vasculature and in mucin-secreting cells and is associated with tissue swelling and a concomitant increase in nasal airway resistance, while the CRTH 2 receptor is expressed on a subset of infiltrating T cells in inflamed nasal mucosa and is associated with the induction of chemotactic migration and/or activation of Th-2, eosinophils, and basophils. 7-9 Collectively, these data support a role for PGD 2 in inflammatory diseases of the upper airways and suggest that blockade of PGD 2 action at either or both receptors might be beneficial for the treatment of nasal allergies and other PGD 2 -mediated inflammatory conditions. This has created significant interest in both DP-1 and CRTH 2 as targets, and several publications have appeared describing inhibitor design. 10,11 PGD 2 is synthesized from arachidonic acid via the oxidation by cyclooxygenase PGH 2 (COX) and isomerization of PGH 2 to PGD 2 by prostaglandin D synthase (PGDS). Hematopoietic PGDS (HPGDS) is responsible for the synthesis of PGD 2 b...

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Calculation of Reduction Potential and p K _a

Jensen

2005

Encyclopedia of Inorganic Chemistry

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Three methods, quantum mechanical and molecular mechanical polarizable continuum model (QM/MM/PCM), QM/PCM, and empirical, were developed and applied to calculate protein reduction potential ( E 0 ) and p K a values. In the QM/MM/PCM calculation of Lys55 p K a of turkey ovomucoid third domain (OMTKY3), the MP2/6‐31 + G(2d,p) method was used to describe the QM region consisting of ∼45 atoms; the rest of the protein was modeled either with ab initio‐derived electric multipole points or atomic charges from standard force field methods; and the aqueous solvation effect was described with the polarizable continuum model (PCM). The calculated Lys55 p K a values are in good agreement with experiments. QM/PCM methods were used to calculate p K a values of various proteins and the E 0 of type‐1 Cu centers. For solvent‐exposed p K a sites, QM/PCM (MP2/6‐31 + G(2d,p) for ∼100 atoms, PCM with dielectric = 78.39 for aqueous solution) methods can often reproduce experimental p K a values. For type‐1 Cu E 0 , because of protein burial, the desolvation effect becomes significant: it was found that Cu‐ligand interactions and desolvation can potentially change the E 0 by ∼300 and ∼400 mV, respectively, using the CPCM/B3LYP/6‐311++G(2df,p) method and model molecules of ∼100 atoms. An empirical method, PROPKA, was developed for protein p K a calculation and analysis, which is able to calculate the p K a values of a protein within ∼1 s.

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Determinants of cysteine pK_a values in creatine kinase and α1‐antitrypsin

Cited by 34 publications

References 25 publications

Exploring the Role of the Active Site Cysteine in Human Muscle Creatine Kinase

Exploring the Role of the Active Site Cysteine in Human Muscle Creatine Kinase

Discovery of an Oral Potent Selective Inhibitor of Hematopoietic Prostaglandin D Synthase (HPGDS)

Calculation of Reduction Potential and p K _a

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Determinants of cysteine pKa values in creatine kinase and α1‐antitrypsin

Cited by 34 publications

References 25 publications

Exploring the Role of the Active Site Cysteine in Human Muscle Creatine Kinase

Exploring the Role of the Active Site Cysteine in Human Muscle Creatine Kinase

Discovery of an Oral Potent Selective Inhibitor of Hematopoietic Prostaglandin D Synthase (HPGDS)

Calculation of Reduction Potential and p K a

Contact Info

Product

Resources

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Determinants of cysteine pK_a values in creatine kinase and α1‐antitrypsin

Calculation of Reduction Potential and p K _a