İnsan serum bütirilkolinesterazının kinetik özellikleri (Normal ve desensitize enzimin kinetik özelliklerinin karşılaştırılması)

Abstract

IV ÖZET İnsan serum bütirilkolinesterazı (açilkolin açilhidrolaz, E.C.3.1.1.8), pH 4.5'da asidik amonyum sülfat kesitlemesi (%55-%70) ve Prokainamid-Sepharose 4B afinite kromatografisi ile 7900 kez saflaştmldı. Enzimin spesifik aktiviîesi 47.6 (imol/dak/mg protein olarak saptandı. Enzim allosterik özelliklere sahip olduğundan ve substratına (bütiriltiyokolin), karşı negatif kooperativite gösterdiğinden, 45°C'da 24 saat ısıtılarak desensitize edilmeye çalışıldı ve enzimin bu işlemle, stabil bir forma dönüştüğü ve kinetik davranışının Michaeiis-Menten kinetiğine uyduğu saptandı. Desensitize enzimin kinetik özellikleri incelenerek, normal enzimin kinetik davranışı ile karşılaştırıldı: a) Normal ve desensitize enzimin kolin, süksinilkolin ve benzoilkolin tarafından inhibe edildiği ve bu subsîrat analoglannın enzimin kısmi kompetitif inhibitörleri olduğu bulundu, b) Ni2+, Co2+ ve Mn2+ iyonlarının her iki enzim formunun lineer karışık tipte inhibitörleri olduğu saptandı, c) Histidini modifiye eden bileşiklerden TLCK ve TPCK'nm aktif merkezde yer alan katalitik üçlüdeki His 438'i modifiye etmedikleri; normal enzimde olduğu gibi, desensitize enzimi de tersinir olarak inhibe ettikleri gösterildi. Fakat, desensitize enzimin substratına, subsîrat analoglanna, metal iyonlarına olan afinitelerinin, normal enzime nazaran daha düşük olduğu saptandı. Anahtar kelimeler: Bütirilkoiinesteraz, insan serumu, desensitizasyon, kinetik özellikler

ABSTRACT Butyrylcholinesterase (acylcholine acylhydrolase, E.C.3.1.1.8) has been purified from human seram by using ammonium sulfate fractionation (55%-70%) with acid step at pH 4.5 and Procainamide-Sepharose 4B affinity chromatography. The overall purification was about 7900 fold and the enzyme had a specific activity of 47.6 (xmol/min/mg protein. Since the enzyme has allosteric behaviour and exhibits negative cooperativity with respect to its substrate (butyrylthiocholine), the purified enzyme was tried to desensitize by heating at 45 °C for 24 hours and it has been observed that, by this procedure the enzyme converts into a stabilized form and follows Michaelis-Menten kinetics. The kinetic properties of the desensitized enzyme was investigated and compared with the kinetic behaviour of the native enzyme: a) The both enzyme (native and desensitized) were inhibited by choline, succinylcholine and benzoilcholine and it has been observed that these substrate analogues are the partial competitive inhibitors of the both forms of the enzyme, b) Native enzyme and the desensitized enzyme could be inhibited by Ni2+, Co2+ and Mn2+ and these metal ions are the linear mixed-type inhibitors of the both type of the enzyme, c) The effects of histidine-modifying reagents on desensitized enzyme were investigated. In the modification studies, it was found that the reagents TLCK and TPCK did not modify the His 438 found in the catalytic triad of the active site, but inhibited the enzyme reversibly as in the case of native enzyme. However, desensitized enzyme has lower affinity to its substrate, substrate analogues, metal ions than that of native enzyme. Key words: Butyrylcholinesterase, human serum, desensitization, kinetic properties