Arg 127 can hydrogen bond the carbonyl oxygen of the

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can interact via hydrogen bonds with the terminal carboxyl group. Arg-127 can hydrogen-bond the carbonyl oxygen of the substrate. All these features have enabled detailed interpretation of many chemical and physico-chemical data at the molecular level. The essential data are as follows: (1) Metal substitution. Table 2.9 lists the divalent metals that have been substituted for zinc (II) in CPA, together with their relative peptidase (and esterase) activities. 22 For some of them, the available x-ray data Table 2.9 Catalytic activities of metal-sub- stituted carboxypeptidases. a 22 Apo Cobalt Nickel Manganese Cadmium Mercury Rhodium Lead Copper Peptidase o 200 50 30 5 o o o Esterase o 110 40 160 140 90 70 60 a Activities are relative to the native enzyme, taken as 100%. b Some activity toward both peptides and esters has recently been observed. 22
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82 2 I THE REACTION PATHWAYS OF ZINC ENZYMES AND RELATED BIOLOGICAL CATALYSTS (2.19) B o -w ~ M ----'" - C I ~ " O .... H-O " H show 123 that the active-site structure is essentially maintained. Even the copper derivative is slightly active. The apoenzyme is completely inac- tive, however. (2) Active-site modifications. Chemical modification and site-directed mu- tagenesis experiments suggest that Glu-270 is essential for cataly- SiS. 124 ,125 Tyr_248,126 Tyr-198,127 and one or more of the arginines 124 are involved but not essential. (3) Kinetics. kcat/K m pH profiles are bell-shaped, characterized by an acid pK, limb around 6 and an alkaline pK a limb around 9: k cat increases with the pK a of 6 and then levels off, and K m increases with a pK a of 9. Several lines of evidence suggest that the pK a = 6 corresponds to the ionization of the Glu-270-coordinated H 2 0 moiety: H I o -c/ M ~ I 0 ... /0 H "H A Site-directed mutagenesis has ruled out Tyr-248 as the group with the pK a of 9 in the rat enzyme. 125,126 Unfortunately, in this enzyme the pK a of 9 is observed in k cat rather than K m ; so the situation for the most- studied bovine enzyme is still unclear. Tyr-248 favors substrate binding three to five times more than the mutagenized Phe-248 derivative. 126 The three possible candidates for this pK, are the coordinated water, Tyr-248, and the metal-coordinated His-196, whose ring NH is not hy- drogen-bonded to any protein residue. 128 The x-ray data at different pH values show a shortening of the Zn-O bond upon increasing pH. 129 This favors the ZnOH hypothesis. (4) Anion binding. The metal binds anionic ligands only below pH 6, i.e., when Glu-270 is protonated, when Glu-270 is chemically 130 or genetically 125 modified, or when aromatic amino acids or related mole- cules are bound in the C-terminal binding domain (Arg-145 + hydro- phobic pocket). 131-134 (5) Intermediates. An anhydride intermediate involving Glu-270 for a slowly hydrolyzed substrate may have been identified. 135 Some other interme- diates have been observed spectroscopically at subzero temperatures with the cobalt(II) derivative.
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