As mentioned above there are a few enzymes in which a

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As mentioned above, there are a few enzymes in which a Ca 2+ ion is pre- sent in the active cleft and essential for activity. Pancreatic phospholipase A 2 (M r = 14 kDa) is an enzyme of this type. The x-ray structure is known to high resolution, and a single Ca 2+ 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 A 2 has been proposed 144 and is shown in Figure 3.30. This mechanism is based on three high-resolution x-ray crystal structures of phospholipase A 2 with and without transition-state analogues bound. The binding constant for Ca 2+ together with the rate of dissociation found from variable-temperature 43Ca NMR studies 145 can be used to calculate k on = 4 X 10 6 M -I S -I, again lower than in EF-hand proteins. Recent IH NMR studies indicate that the global structure of the lipase is very much the same in the Ca 2 +-free and the Ca 2 +-bound forms. Structural changes upon Ca2+ binding appear primarily located in the region of the bind- ing site. 112,146 The mammary glands produce, among other substances, a Ca 2+ -binding enzyme activator, a-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 a- lactalbumin is on the order of 10 7 M -I under physiological conditions. In ad- dition to Ca2+, the enzyme also binds Zn 2+. 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 (M r = 15 kDa) has been
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(A) (B) 153 Figure 3.30 Catalytic mechanism 144 of phospholipase A z . (A) Catalytic attack on substrate bound in a pro- ductive mode. (B) The tetrahedral intermediate as it collapses into products. (C) Products formed by "productive collapse" in which three water molecules move into the active site to replace the products. Two of these water molecules will coordinate the calcium ion. Figure kindly provided by P. B. Sigler.
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154 3 I CALCIUM IN BIOLOGICAL SYSTEMS determined 147 to a high resolution (~1. 7 A). The Ca 2 + -binding site has an in- teresting structure. The ion is surrounded by seven ox·ygen ligands, three from the carboxylate groups of aspartyl residues (82, 87, and 88), two carbonyl oxy- gens (79 and 84), and two water molecules. The spatial arrangement is that of a slightly distorted pentagonal bipyramid with the carbonyl oxygens at the ap- ices, and the five ligands donated by the proteins are part of a tight "elbow"- like tum. The a-lactalbumin site has a superficial structural similarity to an "EF-hand," although the enzyme presumably has no evolutionary relationship with the intracellular Ca 2 + -binding regulatory proteins.
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