Human Tyrosinase: Temperature-Dependent Kinetics of Oxidase Activity

Abstract

Human tyrosinase (Tyr) is involved in pigment biosynthesis, where mutations in its corresponding gene TYR have been linked to oculocutaneous albinism 1, an autosomal recessive disorder. Although the enzymatic capabilities of Tyr have been well-characterized, the thermodynamic driving forces underlying melanogenesis remain unknown. Here, we analyze protein binding using the diphenol oxidase behavior of Tyr and van ’t Hoff temperature-dependent analysis. Recombinant Tyr was expressed and purified using a combination of affinity and size-exclusion chromatography. Michaelis-Menten constants were measured spectrophotometrically from diphenol oxidase reactions of Tyr, using L-3,4-dihydroxyphenylalanine (L-DOPA) as a substrate, at temperatures: 25, 31, 37, and 43 °C. Under the same conditions, the Tyr structure and the L-DOPA binding activity were simulated using 3 ns molecular dynamics and docking. The thermal Michaelis-Menten kinetics data were subjected to the van ‘t Hoff analysis and fitted with the computational model. The temperature-dependent analysis suggests that the association of L-DOPA with Tyr is a spontaneous enthalpy-driven reaction, which becomes unfavorable at the final step of dopachrome formation.

Homology modeling and global computational mutagenesis of human myosin VIIa

“… 27 – 29 One example is tyrosinase with pathogenic mutations causing oculocutaneous albinism type 1A. 30 , 31 Therefore, we hope that biochemical, computational, and clinical validation for a large multi-domain protein like an MYO7A will be possible in the future.…”

The Optimization of anti-tyrosinase activity in extraction of Persicaria pulchra (Bl.) Soják by using Response Surface Methodology (RSM)

“…Mặt khác, nếu có mặt cysteine hoặc glutathione, dopaquinone sẽ chuyển thành cysteinyldopa hoặc glutathionedopa, cuối cùng tạo thành pheomelanin, sắc tố có màu vàng-đỏ 5 . Nhờ có vai trò quan trọng trong hình thành các sắc tố da, nên việc nghiên cứu khả năng ức chế enzyme tyrosinase là cần thiết, góp phần hỗ trợ trong việc điều trị các bệnh lí liên quan đến sắc tố da, hoặc làm trắng da 6 . Nghể (Persicaria pulchra (Bl.)…”

Tyrosinase Nanoparticles: Understanding the Melanogenesis Pathway by Isolating the Products of Tyrosinase Enzymatic Reaction

“…The rate limiting enzyme of the melanogenesis pathway, Tyr, is a 529 amino acid type 1 membrane glycoprotein that consists of an N-terminal signal peptide sequence, EGF-like and tyrosinase domains, and C-terminal transmembrane alpha-helix domain [ 5 ]. Tyr catalyzes the hydroxylation of L-tyrosine into L-3,4-dihydroxyphenylalanine (L-DOPA), which is then oxidized to L-dopaquinone and later dopachrome [ 6 ].…”

“…The full-length protein demonstrated a similar catalytic activity compared to that of the intra-melanosomal tyrosinase domain. Recently, we demonstrated, using van’t Hoff temperature-dependent analysis and Michaelis-Menten kinetics, that the association of L-DOPA with human tyrosinase is a spontaneous enthalpy-driven reaction, which is unfavorable at the final step of dopachrome formation [ 5 ].…”

“…Although unstable due to its ability for self-oxidation, dopachrome is a compound of interest as its production can be used to test for the activity of Tyr while examining its relation to other critical enzymes (Tyrp1 and Tyrp2) in the melanogenesis pathway. Using traditional methods, we previously characterized the enzymatic function of Tyr using the L-DOPA substrate to produce dopachrome and defined its kinetic properties with the presence of protein in solution [ 5 ]. To precisely define parameters of dopachrome reaction within the melanogenesis pathway, the issue of dopachrome isolation from Tyr after enzymatic reaction must be resolved to collect more accurate measurements, which has not been achieved before.…”