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3.5: Extracellular Calcium Ion-binding Proteins

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    The Ca2+ concentration in extracellular fluids is usually orders of magnitude higher than intracellular concentrations. In mammalian body fluids, the "free" Ca2+ concentration is estimated to be 1.25 mM (total Ca2+ is ~2.45 mM) with only minor variations.140 We would thus expect that Ca2+ ions in extracellular fluids playa very different role from that inside cells. To ensure Ca2+ binding the macromolecular binding sites need have only a modest Ca2+ affinity (KBCa2+ ≈ 103 to 104 M-1), and since extracellular Ca2+ does not seem to have a signaling function, the rates of Ca2+ association or dissociation in protein-binding sites need not be very high.

    One particularly important aspect of Ca2+ in mammals is its role in the blood coagulation system. Here we will meet a new type of amino acid, \(\gamma\)-carboxyglutamic acid ("Gla" )-see Figure 3.29, that seems to have been designed by Nature as a Ca2+ ligand with rather special functions. Gla-containing proteins are also encountered in some mineralized tissues. The formation of bone, teeth, and other calcified hard structures is an intriguingly complicated phenomenon that will be dealt with in Section VII. We start, however, with a brief discussion of the role of Ca2+ in some extracellular enzymes.

    Figure 3.29 - Chemical structures of two novel amino acids believed to bind calcium in, e.g., blood-clotting proteins.

    Ca2+-binding in Some Extracellular Enzymes

    Several extracellular enzymes have one or more Ca2+ ions as integral parts of their structure. In a very few of them the Ca2+ ion is bound at or near the active cleft, and appears necessary for maintaining the catalytic activity (phospholipase A2 , \(\alpha\)-amylase, nucleases), whereas other enzymes show catalytic activity even in the absence of Ca2+ (trypsin and other serine proteases). In the latter proteins, the Ca2+ ion is usually ascribed a "structural" role, although its function may be rather more related to "dynamics" and so be more subtle and complex.

    Trypsin has one Ca2+-binding site with four ligands (two side-chain and two backbone oxygens) donated by the protein (Glu-70, Asn-72 , Val-75, and Glu-80) and two ligating water molecules, making the site roughly octahedral.141 The binding constant of Ca2+ to trypsin and its inactive precursor "proenzyme," trypsinogen, has been measured (see Table 3.2). The binding constant is slightly smaller for the precursor, as is also true for chymotrypsin and chymotrypsinogen.142 The Ca2+ affinities of the serine proteases and their proenzymes are such that their Ca2+ sites will be largely occupied in extracellular fluids, but would be unoccupied inside a cell. It has been suggested that this phenomenon constitutes a safeguard against unwanted conversion of the proenzymes into the active enzymes as long as they still are inside the cells where they are synthesized.

    The rates of Ca2+ dissociation of the above enzymes and proenzymes have been measured by 43Ca NMR and stopped-flow techniques,142 and are collected in Table 3.4. We note that the values of kon and koff are generally much smaller than in the intracellular regulatory EF-hand proteins discussed in Section VI. Whereas the latter have dynamic and equilibrium properties similar to those of flexible low-molecular- weight chelators such as EDTA and EGTA, the serine proteases are more similar to the more-rigid cryptates, such as the macrobicyclic amino cryptate [2.2.2] (see Tables 3.2 and 3.4).

    As mentioned above, there are a few enzymes in which a Ca2+ ion is present in the active cleft and essential for activity. Pancreatic phospholipase A2 (Mr ≈ 14 kDa) is an enzyme of this type. The x-ray structure is known to high resolution, and a single Ca2+ ion is found to be surrounded by six ligands, four presented by the protein (Tyr-28, Glu-30, Glu-32, and Asp-49) and two water molecules.143 A mechanism for the action of phospholipase A2 has been proposed144 and is shown in Figure 3.30.


    Figure kindly provided by P. B. Sigler.

    This mechanism is based on three high-resolution x-ray crystal structures of phospholipase A2 with and without transition-state analogues bound. The binding constant for Ca2+ together with the rate of dissociation found from variable-temperature 43Ca NMR studies145 can be used to calculate kon ≈ 4 x 106 M-1s-1, again lower than in EF-hand proteins. Recent 1H NMR studies indicate that the global structure of the lipase is very much the same in the Ca2+-free and the Ca2+-bound forms. Structural changes upon Ca2+ binding appear primarily located in the region of the binding site.112,146

    The mammary glands produce, among other substances, a Ca2+-binding enzyme activator, \(\alpha\)-lactalbumin, that has about 40 percent sequence identity with lysozyme. This protein, which is involved in the conversion of glucose into lactose, is secreted in large quantities, and in human milk constitutes some 15 percent of total protein. The Ca2+-binding constant of bovine or human \(\alpha\)-lactalbumin is on the order of 107 M-1 under physiological conditions. In addition to Ca2+, the enzyme also binds Zn2+. It appears that Ca2+-ion binding affects enzymatic activity, and somehow controls the secretion process, but the biological role of metal-ion binding to a-lactalbumin needs to be studied further. The x-ray structure of a-lactalbumin from baboon milk (Mr ≈ 15 kDa) has been determined147 to a high resolution (~1.7 Å). The Ca2+-binding site has an interesting structure. The ion is surrounded by seven oxygen ligands, three from the carboxylate groups of aspartyl residues (82, 87, and 88), two carbonyl oxygens (79 and 84), and two water molecules. The spatial arrangement is that of a slightly distorted pentagonal bipyramid with the carbonyl oxygens at the apices, and the five ligands donated by the proteins are part of a tight "elbow"-like turn. The \(\alpha\)-lactalbumin site has a superficial structural similarity to an "EF-hand," although the enzyme presumably has no evolutionary relationship with the intracellular Ca2+-binding regulatory proteins.

    Blood clotting proceeds in a complicated cascade of linked events involving many enzymes and proenzymes. About a decade ago it was shown that several of these proteins contained a previously unknown amino acid, \(\gamma\)-carboxyglutamic acid (Gla), and more recently yet another new amino acid, \(\beta\)-hydroxyaspartie acid (Hya), has been discovered (see Figure 3.29). The former is formed postribosomally by a vitamin-K-dependent process in the liver.148 Presently the most-studied Gla protein in the blood-clotting system is prothrombin (Mr ≈ 66 kDa). Ten Gla residues are clustered pairwise in the N-terminal region, essentially lining one edge of the molecule, forming a highly negatively charged region.149 A small (48 residues) proteolytic fragment (F1) that contains all ten Gla amino acids can be prepared. Prothrombin can bind about 10 Ca2+ ions, but F1 binds only 7. Binding studies to F1 show that the Ca2+ ions bind at three high-affinity cooperative sites and four noninteracting sites,150 and that this binding takes places in conjunction with a spectroscopically detectable conformational change (see Table 3.1).

    In the presence of Ca2+ ions, prothrombin and other vitamin-K-dependent proteins in the blood-coagulation system will bind to cell membranes containing acidic phospholipids, in particular, the platelet membrane, which is rich in phosphatidylserine. A proposed model for the prothrombin-membrane interaction is shown in Figure 3.31.

    Figure kindly provided by C. C. F. Blake

    It has long been known that calcium ions are involved in cell-to-cell and cell-to-extracellular matrix interactions, but the molecular details largely remain to be unraveled. In the late 1980s a large, adhesive, calcium-binding matrix glycoprotein (Mr ~ 420 kDa) named thrombospondin was characterized. This multifunctional adhesion molecule is composed of three polypeptide chains, each with 38 amino-acid-Iong repeats that are homologous with the calcium-binding helix-loop-helix sites of the calmodulin superfamily.152 Each thrombospondin molecule is reported to bind 12 calcium ions with an affinity of about 104 M-1, and the removal of calcium is accompanied by a conformational change.153,154

    3.5: Extracellular Calcium Ion-binding Proteins is shared under a CC BY-NC-SA 4.0 license and was authored, remixed, and/or curated by LibreTexts.

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