ID 31866
title alternative
Phosphodiester Hydrolysis by a New Zinc(II) Macrocyclic Tetraamine Complex with an Alcohol Pendant : Elucidation of the Roles of Ser-102 and Zinc(II) in Alkaline Phosphatase
Kodama, Yorimitsu
Medical sciences


6)BNPと亜鉛錯体とから中間体が生成する反応をpH6-10で検討した。得られたシグモイド曲線から速度論的酸解離定数は7.4となり、pH滴定法から求めた亜鉛錯体の酸解離定数7.3とほぼ一致した。この結果、この反応の活性種はアルコキシド型錯体であると結論された。この反応の2次反応速度定数は6.5×104M1s-1と決定され、ZnII-methylcyclenによるBNPの加水分解反応速度定数5.2×10-6M-1s-1の125倍にもなり、ZnII--ORはZnII-OH-よりも強い求核体であることがわかった。7)中間体から最終生成物へのリン酸ジエステルぺンダント加水分解反応をpH6-10で検討した。得られたシグモイド曲線から速度論的酸解離定数は9.0となり、pH滴定法から求めた中間体の亜鉛イオンの配位水の酸解離定数9.1とほぼ一致した。この結果、この反応の活性種は分子内ZnII-OH-であると結論された。この反応の1次反応速度定数は3.5×10-5s-1と決定した。8)同様の加水分解反応が分子間で起きた場合のモデルとしてZnII-methylcyclenと4-ニトロフェニル エチル リン酸との反応(2次反応速度定数:7.9×10-7M-1s-1)を考えると、速度定数の比較から、リン酸ジエステルと亜鉛錯体との分子間反応が分子内で起きることによる有効モル濃度は45Mとなった。この結果、基質を求核体の非常に近い位置に固定することによって反応性が上がることがわかった。

A new benzyl alcohol-pendant cyclen (cyclen = 1,4,7, 10-tetraazacyclododecane) ligand, (S)-1 -(2-hydroxy-2-phenylethyl)-1,4,7,10-tetraazacyclododecane (L) has been synthesized. The complexation of L with ZnII yielded 1:1 five-coordinate complexes (isolated as its perchlorate salts with the pendant alcohol either undissociated (ZnL) or dissociated (ZnH-1L) from acidic (pH 6.0) or basic (pH 9.5) aqueous solution, respectively). The pKa value for the pendant alcohol (ZnL ⇌ ZnH-1L + H+) was determined by potentiometric pH titration to be 7.30 ± 0.02 at 35 °C with l = (1. 10 (NaNO3). The X-ray crystal study of ZnH-1L has shown two crystallographically distinct structures with the alkoxide closely coordinated at the fifth coordination site, where an average distance of Zn-O is 1.91 Å. The ZnII-bound alkoxide anion in ZnH-1L is a more reactive nucleophile than N-methylcyclen-ZnII-OH species (ZnII-Me-cyclen). In the kinetic study using ZnL in aqueous solution (pH 6.0-10.3) at 35 °C with l = 0. 10 (NaNO3), the rate-pH profile for a phosphoryl transfer reaction from bis(4-nitrophenyl)phosphate (BNP-) to ZnH-1L gave a sigmoidal curve with an inflection point at pH 7.4, which corresponds to the pKa value for ZnL ⇌ ZnH-1L + H+. The second-order rate constant kBNP of (6.5 ± 0.1) x 10-1 M-1 s-1 is 125 times greater than the corresponding value of (5.2 ± 0.2) x 10-6 M-1 s-1 for BNP- hydrolysis catalyzed by ZnII-Me-cyclen. The product of the phosphoryl transfer reaction from BNP- to ZnH-1L is the pendant alcoholphosphorylated ZnH-1L-NPP, which was isolated as its perchlorate salts ZnL-NPP by reacting ZnH-1L with BNP- in DMF. In anhydrous DMF solution, the phosphoryl transfer (kBNP of 1.1 ± 0.1 M-1 s-1 at 35 °C) is 1700 times faster than that in aqueous solution.

In the subsequent reaction of ZnL-NPP, the pendant phosphodiester undergoes an intramolecular nucleophilic attack by the ZnII-bound OH of ZnH-1L-NPP to yield a phosphomonoester product ZnL-P. From the sigmoidal rate-pH relationship (pH 7.4-10.5), the kinetic pKa value of 9.0 was estimated for ZnL-NPP ⇌ ZnH-1L-NPP + H+, which is almost the same value (pKa = 9.10 ± 0.05) determined by potentiometric pH titration at 35 °C. The first-order rate constant for the reaction ZnH-1L-NPP → ZnL-P is (3.5 ± 0. 1) x 10-5 s-1 at 35 °C with l = 0.10 (NaNO3). As a reference to this intramolecular phosphodiester hydrolysis, ethyl (4-nitrophenyl) phosphate (NEP-) was hydrolyzed by ZnII-Me-cyclen. The second-order rate constant kNEP was (7.9 ± 0.3) x 10-7 M-1 s-1 at 35 °C with l = 0. 10 (NaNO3). Thus, the intramolecular hydrolysis is 45,000 times faster than the intermolecular NEP hydrolysis with 1 mM ZnII-Me-cyclen. The present findings that demonstrate the potential of the proximate alcohol by ZnII in the initial phosphoryl transfer and the potential of the ZnII-bound water in the intramolecular phosphate hydrolysis may well serve to elucidate the collaborative functions of Ser-102 and ZnII ions in alkaline phosphatase.
Abstract / p1
1.Introduction / p2
2.Results and Discution / p5
 2-1.Synthesis of Benzyl Alcohol-Pendant Cyclen / p5
 2-2.Protonation and Zinc(II)Complexation Constants of the Benzyl Alcohol-Pendant Cyclen / p5
 2-3.X-ray Crystal Structure of the ZnII Complex of the Deprotonated Benzyl Alcohol-Pendant Cyclen / p7
 2-4.Net Reaction of the Benzyl Alcohol-Pendant Cyclen-ZnII Complex with Bis(4-nitrophenyl)Phosphate / p10
 2-5.A Kinetic Study of the Initial Reaction / p12
 2-6.Isolation of the Phosphoryl Intermediate / p14
 2-7.Spontaneous Hydrolysis of the Pendant Phosphodiester in the Phosphoryl Intermediate to a Phosphomonoester by the Intramolecular ZnII-OH⁻ / p14
 2-8.Hydrolysis of Ethyl(4-Nitrophenyl)Phosphate with an Intermolecular Nucleophile ZnII-OH⁻ / p16
3.Summary and Conclusions / p17
4.Experimental Section / p18
References and Notes / p23
Acknowledgement / p25
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広島大学(Hiroshima University)
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Graduate School of Biomedical Science