Publication: Monoamin oksidaz b (MAOB) katalizleme tepkimesine aktif bölgenin etkisi
Abstract
MONOAMİN OKSİDAZ B (MAO B) KATALİZLEME TEPKİMESİNE AKTİF BÖLGENİN ETKİSİ Mitokondrial monoamin oksidaz flavin içeren ve nörotransmitter aminlerin oksidasyonunu katalizleyen bir enzimdir. Substrat ve inhibitör seçiciliklerinde farklılıklar gösteren MAO A ve MAO B olarak bilinen iki formu vardır. Bu enzimlerin inhibitörleri depresyon, Parkinson, Alzeimer gibi hastalıkların tedavisinde kullanıldığından, amin oksitleme mekanizmalarının detaylarının bilinmesi büyük önem arzetmektedir. Bu çalışmada, MAO B aktif bölgesinin amin oksitleme mekanizmasına etkisi kuantum mekanik PM6 ve/ veya ONIOM(M06-2X/ 6-13+G(d,p):PM6) yöntemleri ile çalışıldı. İki-basamaklı hidrür transferi mekanizması ve polar nükleofilik mekanizma benzilamin ve aktif bölgedeki Tyr188, Tyr398, Tyr435 ve 7 su molekülü ile birlikte modellendi. Çıktılar dört madde ile özetlenebilir: 1) Literatürde önerilen N5-araürünün hidrür transferi sırasında oluşma zorunluluğunun olmadığı gösterilmiştir. Onun yerine ürün kompleksi oluşmaktadır ve bu kompleks N5-araürün ile dengeye gelebilir. Böylece, düzeltilmiş yeni bir hidrür mekanizması önerilmiştir. 2) Literatürde önerilen polar nükleofilik mekanizma da revize edilerek, üç basamaktan oluşan yeni bir mekanizma önerilmiştir. 3) Aktif bölgedeki Tyr398 ve Tyr435’in aktivasyon enerjisi üzerine etkisi Tyr398X ve Tyr435X mutant modelleri (X=Phe, His, Trp, Leu) üzerinden incelenmiştir. Tyr398X ile substrat arasındaki CH/ π çekimlerinin katalizleme tepkimesinde önemli olabileceği tespit edilmiştir. 4) Farklı büyüklüklerde beş model yapı kullanarak kuantum kümeleme yaklaşımı uygulanmıştır. Model I, II, III, IV, V yapıları kademeli olarak büyürken aktivasyon enerji değişimi karşılaştırılmıştır. En çarpıcı değişim, Model IV’te flavine kovalent bağlı olan Cys397’nin yapıya dahil edilmesi ile ortaya çıkmıştır. Model V’in aktivasyon enerjisinde ise önemli bir değişim olmamıştır. Böylece, MAO enzimlerinin aktif bölgesini temsil edebilecek optimum yapı olarak Model IV önerilmiştir.
THE EFFECT OF ACTIVE SITE ON MONOAMIN OXIDASE (MAO B) CATALYSIS REACTION Mitochondrial monoamine oxidase is a flavoenzyme which catalyzes the oxidation of neurotransmitter amines. The two forms of the enzyme, know as MAO A and MAO B, have different selectivities for their substrates and inhibitors. MAO inhibitors are used to cure depression, Parkinson, Alzheimer etc. Therefore, understanding the details of their amine oxidation mechanism has an outmost significance. In this study, the effect of enzyme active site on the amine oxidation mechanism of MAO B was studied with quantum mechanics PM6 and/ or ONIOM(M06-2X/ 6-13+G(d,p):PM6) methods. Two step hydride transfer mechanism and polar nucleophilic B mechanism were modelled by using benzylamine and MAO B including Tyr188, Tyr398, Tyr435 and 7 water molecules in the active site. The outcome can be summarized in four parts: 1) It was shown that the proposed flavin N5-adduct does not necessarily form during the hydride transfer reaction. Instead, the product complex forms, which can be in equilibrium with the N5-adduct. Thus, we proposed a modified hydride transfer mechanism. 2) A modified polar nucleophilic mechanism involving three steps was also proposed. 3) The effect of Tyr398 and of Tyr435 on the activation energy barrier were investigated by means of Tyr398X and Tyr435X mutant models where X=Phe, His, Trp, Leu. It was shown that favorable CH/ π interactions between Tyr398X aromatic π-electrons and substrate play an important role in catalysis. 4) Quantum cluster approach was used employing five different model structures with different sizes of the active site. The activation energies were compared with respect to the gradually increasing sizes of the Models I, II, III, IV, V. The striking decrease in activation energy was observed when covalently bonded Cys397 was included in the active site model in Model IV, and activation energy of Model V did not change considerably. Thus, we propose Model IV as the optimum model representing the active sites for MAO enzymes.
THE EFFECT OF ACTIVE SITE ON MONOAMIN OXIDASE (MAO B) CATALYSIS REACTION Mitochondrial monoamine oxidase is a flavoenzyme which catalyzes the oxidation of neurotransmitter amines. The two forms of the enzyme, know as MAO A and MAO B, have different selectivities for their substrates and inhibitors. MAO inhibitors are used to cure depression, Parkinson, Alzheimer etc. Therefore, understanding the details of their amine oxidation mechanism has an outmost significance. In this study, the effect of enzyme active site on the amine oxidation mechanism of MAO B was studied with quantum mechanics PM6 and/ or ONIOM(M06-2X/ 6-13+G(d,p):PM6) methods. Two step hydride transfer mechanism and polar nucleophilic B mechanism were modelled by using benzylamine and MAO B including Tyr188, Tyr398, Tyr435 and 7 water molecules in the active site. The outcome can be summarized in four parts: 1) It was shown that the proposed flavin N5-adduct does not necessarily form during the hydride transfer reaction. Instead, the product complex forms, which can be in equilibrium with the N5-adduct. Thus, we proposed a modified hydride transfer mechanism. 2) A modified polar nucleophilic mechanism involving three steps was also proposed. 3) The effect of Tyr398 and of Tyr435 on the activation energy barrier were investigated by means of Tyr398X and Tyr435X mutant models where X=Phe, His, Trp, Leu. It was shown that favorable CH/ π interactions between Tyr398X aromatic π-electrons and substrate play an important role in catalysis. 4) Quantum cluster approach was used employing five different model structures with different sizes of the active site. The activation energies were compared with respect to the gradually increasing sizes of the Models I, II, III, IV, V. The striking decrease in activation energy was observed when covalently bonded Cys397 was included in the active site model in Model IV, and activation energy of Model V did not change considerably. Thus, we propose Model IV as the optimum model representing the active sites for MAO enzymes.