Ein L³ZnOH‐Komplex als funktionelles Modell des Enzyms Carboanhydrase

Abstract

An L3ZnOH Complex as a Functional Model of the Enzyme Carbonic AnhydraseThe tridentate ligand hydrotris(3-tert-butyl-5-methylpyrazo-1yl)borate (L3) reacts with zinc perchlorate hexahydrate to form L3ZnOH (1) which is the first mononuclear neutral zinc hydroxide complex. 1 is a structural model of the enzyme carbonic anhydrase due to its tetrahedral coordination with three N-heterocycles and one OH ion as ligands. Its functional analogy to the enzyme involves (a) reversible uptake of COz forming the unstable bicarbonate complex L3ZnOCOOH (4) in solution from which the dinuclear carbonate complex L3ZnOC(0)OZnL3 (3) crystallizes, (b) reaction with dialkyl pyrocarbonates forming the complexes L3ZnOC(0)OR (5) which are stable esters of the bicarbonate complex and which are easily hydrolyzed to ROH, COz, and L3ZnOH, (c) thermal decarboxylation of the ethyl carbonate complex 5b resulting in the ethanolate complex L3ZnOEt (6) which is extremely sensitive to hydrolysis, (d) "inhibition" by small anions due to conversion of 1 into the complexes L3ZnX (?: X = C1, CN, N3, OAc). The identity of the various L3Zn derivatives is established by NMR methods and structure determinations. Carboanhydrase ist eines der am besten untersuchten Enzyme[']. Ihre bemerkenswert einfache Funktion besteht in der Katalyse der hydrolytischen Umwandlung zwischen Kohlendioxid und Hydrogencarbonat, die normalerweise langsam, da kinetisch gehemmt, ist. An vielen Stellen im Korper, so auch in den roten Blutkorperchen als das biologische Gegenstuck des Hamoglobins, sorgt sie damit fur den raschen Umsatz bzw. die rasche Entfernung des korpereigenen Verbrennungsprodukts C02. Ihr katalytisch aktives Zentrum besteht aus einem Zink-Ion, das tetraedrisch von drei Histidin-Imidazol-Gruppen und einem Wassermolekul umgeben ist. Als Teilschritte des Katalysecyclus werden die Deprotonierung des H20-Liganden und der Angriff des starken Nucleophils [His3ZnOH] + an C 0 2 unter Bildung eines Hydrogencarbonat-Liganden diskutiert, der dann unter Substitution durch ein neues Wassermolekiil abgelost wird[',21. Da jedoch bis jetzt weder der Enzym-Substrat-(C02)-Komplex noch andere Intermediate des katalytischen Cyclus durch Strukturanalysen identifiziert sind, beruhen die Vorschlage zu seinem Verlauf bisher nur auf kinetischen, spektroskopischen und theoretischen StudienL3]. Ansatze zur strukturellen Modellierung des Enzyms konnten in der Vergangenheit nicht die tetraedrische N3-ZnOH2-Koordination reprod~zieren[~], und Studien zur funktionellen Modellierung rnit anderen Zink-Komplexen blieben zumeist auf Ester-Hydrolysen beschrankt [' I. Die beste Annaherung an den Zustand im Enzym gelang bisher Kimura et al. rnit dem Komplex [(1,5,9-Triazacyclododecan)ZnOH2I2+, der nahe dem Neutralpunkt deprotoniert wird und die CO,-Hydratisierung katalysiert [61. Einen ein deutig charakterisierten einkernigen tetraedrischen L3ZnOH-Komplex gab es zu Beginn unserer Untersuchungen nicht, obgleich basische Salze eine normale Erscheinung in der anorganischen Chemie von Zink sind"] und obgle...

Intrinsic Proton-Donating Power of Zinc-Bound Water in a Carbonic Anhydrase Active Site Model Estimated by NMR

“…The present study was devoted to neutral tris(pyrazolyl)hydroborate zinc hydroxide complexes of the type [Tp R1,R2 ZnOH] designed by Vahrenkamp and Trofimenko. 31,32,33,34 The advantage of these complexes is that they are soluble in some polar organic solvents which seems to better mimic the electrostatic properties of enzymic acid sites than aqueous solutions. 28 As these complexes are not soluble in water, their p K a values could not be measured directly but only estimated to be smaller than 7.…”

Biomimetic Applications of Metal Systems Supported by Scorpionates

“…The most difficult task, which is not fully achieved as yet, is the isolation of a {[HB(pz R ) 3 ]Zn(OH 2 )} + cation Bergquist & Parkin, 1999) ([HB(pz R ) 3 ] denotes any one substituted tris(pyrazolyl)borate ligand), the main reason for which is the hydrolytic destruction of the [HB(pz R ) 3 ] -ligands in acidic media. In all cases studied, the [HB(pz R ) 3 ]Zn-OH complexes are the easiest to prepare (Alsfasser et al, 1993;. In just a few cases they could be induced to release water and form the dinuclear complexes {[HB(pz R ) 3 ]Zn-OH-Zn[HB(pz R ) 3 ]} + ; full deprotonation with formation of the neutral molecular species {[HB(pz R ) 3 ]Zn-OZn[HB(pz R ) 3 ]} have been achieved in one case .…”

Evidence for a Trajectory of Hydrolytic Reactions brought about by [L3Zn−OH] Species

“…The [Tp'ZnÀOH] complexes have also been found to hydrolyze pyrophosphates, [49] amides, [47] and CO 2 . [45,46] The mechanistic details of pyrophosphate cleavage should correspond to those of organophosphate cleavage, and the details of amide, peptide, and CO 2 hydrolysis to those of ester hydrolysis. Viewed from the Tp'Zn unit the atomic motions during the reactions are equivalent in all cases.…”

“…[42,43] We have contributed, to this field, the synthesis of the stable pyrazolylborate ± zinc hydroxide complexes 1 ± 5, [14,16,18,19] experimental proof that the pK a values of the corresponding ZnÀOH 2 complexes are at or below 7, [44] and the hydrolytic reactions of these complexes with CO 2 , [45,46] esters and amides, [47] organophosphates, [48] diphosphates, [49] and sugar phosphates including nucleotide derivatives. [50] In a short communication Kitajima described similar organophosphate cleavages by another [Tp'ZnÀOH] complex.…”