{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,2,19]],"date-time":"2026-02-19T03:36:58Z","timestamp":1771472218205,"version":"3.50.1"},"reference-count":31,"publisher":"MDPI AG","issue":"15","license":[{"start":{"date-parts":[[2021,7,21]],"date-time":"2021-07-21T00:00:00Z","timestamp":1626825600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000009","name":"Foundation for the National Institutes of Health","doi-asserted-by":"publisher","award":["ZIA EY000476-10"],"award-info":[{"award-number":["ZIA EY000476-10"]}],"id":[{"id":"10.13039\/100000009","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Human tyrosinase (Tyr) is a glycoenzyme that catalyzes the first and rate-limiting step in melanin production, and its gene (TYR) is mutated in many cases of oculocutaneous albinism type 1 (OCA1). The mechanisms by which individual mutations contribute to the diverse pigmentation phenotype in patients with OCA1 have only began to be examined and remain to be delineated. Here, we analyze the temperature-dependent kinetics of wild-type Tyr (WT) and two OCA1B mutant variants (R422Q and P406L) using Michaelis\u2013Menten and Van\u2019t Hoff analyses. Recombinant truncated human Tyr proteins (residues 19\u2013469) were produced in the whole insect Trichoplusia Ni larvae. Proteins were purified by a combination of affinity and size-exclusion chromatography. The temperature dependence of diphenol oxidase protein activities and kinetic parameters were measured by dopachrome absorption. Using the same experimental conditions, computational simulations were performed to assess the temperature-dependent association of L-DOPA and Tyr. Our results revealed, for the first time, that the association of L-DOPA with R422Q and P406L followed by dopachrome formation is a complex reaction supported by enthalpy and entropy forces. We show that the WT has a higher turnover number as compared with both R422Q and P406L. Elucidating the kinetics and thermodynamics of mutant variants of Tyr in OCA1B helps to understand the mechanisms by which they lower Tyr catalytic activity and to discover novel therapies for patients.<\/jats:p>","DOI":"10.3390\/ijms22157771","type":"journal-article","created":{"date-parts":[[2021,7,21]],"date-time":"2021-07-21T11:53:23Z","timestamp":1626868403000},"page":"7771","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":9,"title":["Characterization of Temperature-Dependent Kinetics of Oculocutaneous Albinism-Causing Mutants of Tyrosinase"],"prefix":"10.3390","volume":"22","author":[{"given":"Samuel A.","family":"Wachamo","sequence":"first","affiliation":[{"name":"National Eye Institute, National Institutes of Health, Bethesda, MD 20892, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Milan H.","family":"Patel","sequence":"additional","affiliation":[{"name":"National Eye Institute, National Institutes of Health, Bethesda, MD 20892, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Paul K.","family":"Varghese","sequence":"additional","affiliation":[{"name":"National Eye Institute, National Institutes of Health, Bethesda, MD 20892, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Monika B.","family":"Dolinska","sequence":"additional","affiliation":[{"name":"National Eye Institute, National Institutes of Health, Bethesda, MD 20892, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7204-6572","authenticated-orcid":false,"given":"Yuri V.","family":"Sergeev","sequence":"additional","affiliation":[{"name":"National Eye Institute, National Institutes of Health, Bethesda, MD 20892, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2021,7,21]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"41","DOI":"10.1111\/pcmr.12546","article-title":"Oculocutaneous albinism type 1: Link between mutations, tyrosinase conformational stability, and enzymatic activity","volume":"30","author":"Dolinska","year":"2017","journal-title":"Pigment Cell Melanoma Res."},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Dolinska, M.B., Kovaleva, E., Backlund, P., Wingfield, P.T., Brooks, B.P., and Sergeev, Y.V. (2014). Albinism-causing mutations in recombinant human tyrosinase alter intrinsic enzymatic activity. PLoS ONE, 9.","DOI":"10.1371\/journal.pone.0084494"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"320","DOI":"10.1034\/j.1600-0749.2000.130503.x","article-title":"The tyrosinase gene and oculocutaneous albinism type 1 (OCA1): A model for understanding the molecular biology of melanin formation","volume":"13","author":"Oetting","year":"2000","journal-title":"Pigment Cell Res."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"1143","DOI":"10.1016\/j.jid.2018.10.033","article-title":"The Molecular Basis of Chemical Chaperone Therapy for Oculocutaneous Albinism Type 1A","volume":"139","author":"Teramae","year":"2019","journal-title":"J. Investig. Dermatol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"1046","DOI":"10.1172\/JCI115064","article-title":"Temperature-sensitive tyrosinase associated with peripheral pigmentation in oculocutaneous albinism","volume":"87","author":"King","year":"1991","journal-title":"J. Clin. Investig."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"23707","DOI":"10.1016\/S0021-9258(18)35895-2","article-title":"Mutational mapping of the catalytic activities of human tyrosinase","volume":"267","author":"Tripathi","year":"1992","journal-title":"J. Biol. Chem."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"259","DOI":"10.1042\/bj3550259","article-title":"The molecular basis of oculocutaneous albinism type 1 (OCA1): Sorting failure and degradation of mutant tyrosinases results in a lack of pigmentation","volume":"355","author":"Toyofuku","year":"2001","journal-title":"Biochem. J."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"1383","DOI":"10.1136\/bjo.2005.070243","article-title":"Temperature sensitive oculocutaneous albinism associated with missense changes in the tyrosinase gene","volume":"89","author":"Wang","year":"2005","journal-title":"Br. J. Ophthalmol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"13428","DOI":"10.1021\/acs.jmedchem.0c00994","article-title":"Advances in the Design of Genuine Human Tyrosinase Inhibitors for Targeting Melanogenesis and Related Pigmentations","volume":"63","author":"Roulier","year":"2020","journal-title":"J. Med. Chem."},{"key":"ref_10","doi-asserted-by":"crossref","unstructured":"Varghese, P.K., Abu-Asab, M., Dimitriadis, E.K., Dolinska, M.B., Morcos, G.P., and Sergeev, Y.V. (2021). Tyrosinase Nanoparticles: Understanding the Melanogenesis Pathway by Isolating the Products of Tyrosinase Enzymatic Reaction. Int. J. Mol. Sci., 22.","DOI":"10.3390\/ijms22020734"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"81","DOI":"10.15406\/japlr.2020.09.00356","article-title":"Functional in silico analysis of human tyrosinase and OCA1 associated mutations","volume":"9","author":"Patel","year":"2020","journal-title":"J. Anal. Pharm. Res."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"520","DOI":"10.1111\/pcmr.12393","article-title":"Melanins and melanogenesis: From pigment cells to human health and technological applications","volume":"28","author":"Wakamatsu","year":"2015","journal-title":"Pigment Cell Melanoma Res."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"E8","DOI":"10.1038\/skinbio.2011.4","article-title":"Determination of melanin synthetic pathways","volume":"131","author":"Hearing","year":"2011","journal-title":"J. Investig. Dermatol."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"374","DOI":"10.1111\/1523-1747.ep12680867","article-title":"Peroxidatic conversion of tyrosine to dopachrome","volume":"63","author":"Patel","year":"1974","journal-title":"J. Investig. Dermatol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"3","DOI":"10.1111\/j.1600-0749.2005.00288.x","article-title":"Tyrosinase maturation through the mammalian secretory pathway: Bringing color to life","volume":"19","author":"Wang","year":"2006","journal-title":"Pigment Cell Res."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"6.15.1","DOI":"10.1002\/cpps.37","article-title":"Purification of Recombinant Human Tyrosinase from Insect Larvae Infected with the Baculovirus Vector","volume":"89","author":"Dolinska","year":"2017","journal-title":"Curr. Protoc. Protein Sci."},{"key":"ref_17","doi-asserted-by":"crossref","unstructured":"Young, K.L., Kassouf, C., Dolinska, M.B., Anderson, D.E., and Sergeev, Y.V. (2020). Human Tyrosinase: Temperature-Dependent Kinetics of Oxidase Activity. Int. J. Mol. Sci., 21.","DOI":"10.3390\/ijms21030895"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"2149","DOI":"10.1096\/fj.01-0216com","article-title":"Oculocutaneous albinism types 1 and 3 are ER retention diseases: Mutation of tyrosinase or Tyrp1 can affect the processing of both mutant and wild-type proteins","volume":"15","author":"Toyofuku","year":"2001","journal-title":"FASEB J."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"840","DOI":"10.1007\/BF02980030","article-title":"Temperature regulates melanin synthesis in melanocytes","volume":"26","author":"Kim","year":"2003","journal-title":"Arch. Pharm. Res."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"1719","DOI":"10.1038\/s41598-017-19135-7","article-title":"Effects of Distal Mutations on the Structure, Dynamics and Catalysis of Human Monoacylglycerol Lipase","volume":"8","author":"Tyukhtenko","year":"2018","journal-title":"Sci. Rep."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/j.sbi.2018.09.004","article-title":"Modulation of allosteric coupling by mutations: From protein dynamics and packing to altered native ensembles and function","volume":"54","author":"Naganathan","year":"2019","journal-title":"Curr. Opin. Struct. Biol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"20.13.1","DOI":"10.1002\/0471140864.ps2013s77","article-title":"Thermodynamic analysis of weak protein interactions using sedimentation equilibrium","volume":"77","author":"Sergeev","year":"2014","journal-title":"Curr. Protoc. Protein. Sci."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"3096","DOI":"10.1021\/bi00514a017","article-title":"Thermodynamics of protein association reactions: Forces contributing to stability","volume":"20","author":"Ross","year":"1981","journal-title":"Biochemistry"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"481","DOI":"10.1046\/j.1523-1747.2002.01824.x","article-title":"Coexpression of wild-type tyrosinase enhances maturation of temperature-sensitive tyrosinase mutants","volume":"119","author":"Halaban","year":"2002","journal-title":"J. Investig. Dermatol."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"1984","DOI":"10.1271\/bbb.68.1984","article-title":"Tyrosinase inhibitor isolated from the leaves of Zanthoxylum piperitum","volume":"68","author":"Jeong","year":"2004","journal-title":"Biosci. Biotechnol. Biochem."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"2241","DOI":"10.1099\/mic.0.2007\/006833-0","article-title":"Involvement of a novel copper chaperone in tyrosinase activity and melanin synthesis in Marinomonas mediterranea","volume":"153","author":"Solano","year":"2007","journal-title":"Microbiology"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"37298","DOI":"10.1038\/srep37298","article-title":"In silico Mapping of Protein Unfolding Mutations for Inherited Disease","volume":"6","author":"McCafferty","year":"2016","journal-title":"Sci. Rep."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"W382","DOI":"10.1093\/nar\/gki387","article-title":"The FoldX web server: An online force field","volume":"33","author":"Schymkowitz","year":"2005","journal-title":"Nucleic Acids Res."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"114","DOI":"10.1002\/prot.22570","article-title":"Improving physical realism, stereochemistry, and side-chain accuracy in homology modeling: Four approaches that performed well in CASP8","volume":"77","author":"Krieger","year":"2009","journal-title":"Proteins"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"2981","DOI":"10.1093\/bioinformatics\/btu426","article-title":"YASARA View\u2014Molecular graphics for all devices\u2014From smartphones to workstations","volume":"30","author":"Krieger","year":"2014","journal-title":"Bioinformatics"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"455","DOI":"10.1002\/jcc.21334","article-title":"AutoDock Vina: Improving the speed and accuracy of docking with a new scoring function, efficient optimization and multithreading","volume":"31","author":"Trott","year":"2010","journal-title":"J. Comput. Chem."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/22\/15\/7771\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T06:32:40Z","timestamp":1760164360000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/22\/15\/7771"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,7,21]]},"references-count":31,"journal-issue":{"issue":"15","published-online":{"date-parts":[[2021,8]]}},"alternative-id":["ijms22157771"],"URL":"https:\/\/doi.org\/10.3390\/ijms22157771","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,7,21]]}}}