Human Tyrosinase: Temperature-Dependent Kinetics of Oxidase Activity

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.

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Characterization of Temperature-Dependent Kinetics of Oculocutaneous Albinism-Causing Mutants of Tyrosinase

International Journal of Molecular Sciences ◽

10.3390/ijms22157771 ◽

2021 ◽

Vol 22 (15) ◽

pp. 7771

Author(s):

Samuel A. Wachamo ◽

Milan H. Patel ◽

Paul K. Varghese ◽

Monika B. Dolinska ◽

Yuri V. Sergeev

Keyword(s):

Trichoplusia Ni ◽

Oculocutaneous Albinism ◽

Size Exclusion ◽

Temperature Dependent ◽

Experimental Conditions ◽

Rate Limiting Step ◽

Its Gene ◽

Exclusion Chromatography ◽

First Time ◽

Kinetics Of

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 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–Menten and Van’t Hoff analyses. Recombinant truncated human Tyr proteins (residues 19–469) 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.

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Mechanism and kinetics of inactivation at 40–70°C of the extracellular proteinase from Pseudomonas fluorescens 22F

Journal of Dairy Research ◽

10.1017/s0022029997002719 ◽

1998 ◽

Vol 65 (2) ◽

pp. 261-272 ◽

Cited By ~ 11

Author(s):

ERIX P. SCHOKKER ◽

MARTINUS A. J. S. VAN BOEKEL

Keyword(s):

Molecular Mass ◽

Pseudomonas Fluorescens ◽

Computer Simulations ◽

Rate Constants ◽

Size Exclusion ◽

Temperature Dependent ◽

Extracellular Proteinase ◽

Reaction Rate Constants ◽

Exclusion Chromatography ◽

Kinetics Of

HPLC size exclusion chromatography experiments showed that during inactivation at 40–70°C of the extracellular proteinase from Pseudomonas fluorescens 22F small molecular mass fragments were formed, indicating that autoproteolysis was at least one of the major causes of inactivation. The formation of small molecular mass fragments and the reaction order indicated that intermolecular autoproteolysis was more likely than intramolecular autodigestion. This was confirmed by computer simulations. The rate constants and the activation enthalpy (ΔH[Dagger]) and entropy (ΔS[Dagger]) for the reactions of the intermolecular autoproteolysis model were derived from computer simulations. ΔH[Dagger] and ΔS[Dagger] of the unfolding reaction were 504 kJ mol−1 and 1252 J mol K−1 respectively. ΔH[Dagger] and ΔS[Dagger] of the refolding reaction were strongly temperature dependent. The estimates for the enthalpy (ΔH0) and entropy (ΔS0) difference between the folded and unfolded state as derived from the reaction rate constants of unfolding and refolding were subject to large deviations, owing to accumulation of errors in the estimation of the kinetic characteristics.

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Kinetic and structural analysis of the ultrasensitive behaviour of cyanobacterial ADP-glucose pyrophosphorylase

Biochemical Journal ◽

10.1042/bj3500139 ◽

2000 ◽

Vol 350 (1) ◽

pp. 139-147 ◽

Cited By ~ 11

Author(s):

Diego F. GÓMEZ CASATI ◽

Miguel A. AON ◽

Alberto A. IGLESIAS

Keyword(s):

Conformational Changes ◽

Tertiary Structure ◽

Fluorescence Emission ◽

Maximal Activity ◽

Size Exclusion ◽

Exclusion Chromatography ◽

Adp Glucose Pyrophosphorylase ◽

Kinetics Of ◽

Allosteric Regulators

The kinetic and (supra)molecular properties of the ultrasensitive behaviour of ADP-glucose pyrophosphorylase (AGPase) from Anabaena PCC 7120 (a cyanobacterium) were exhaustively studied. The response of the enzyme toward the allosteric activator 3-phosphoglycerate (3PGA) occurs with ultrasensitivity as a consequence of the cross-talk with the inhibitor Pi. Molecular ‘crowding’renders AGPase more sensitive to the interplay between the allosteric regulators and, consequently, enhances the ultrasensitive response. In crowded media, and when orthophosphate is present, the activation kinetics of the enzyme with 3PGA proceed with increased co-operativity and reduced affinity toward the activator. Under conditions of ultrasensitivity, the enzyme's maximal activation takes place in a narrow range of 3PGA concentrations. Moreover, saturation kinetics of the enzyme with respect to its substrates, glucose 1-phosphate and ATP, were different at low or high 3PGA levels in crowded media. Only under the latter conditions did AGPase exhibit discrimination between low or high levels of the activator, which increased the affinity toward the substrates and the maximal activity reached by the enzyme. Studies of fluorescence emission of tryptophan residues, fourth-derivative spectroscopy and size-exclusion chromatography indicated that the ultrasensitive behaviour is correlated with intramolecular conformational changes induced in the tertiary structure of the homotetrameric enzyme. The results suggest a physiological relevance of the ultrasensitive response of AGPase in vivo, since the enzyme could be subtly sensing changes in the levels of allosteric regulators and substrates, and thus determining the flux of metabolites toward synthesis of storage polysaccharides.

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The Two ADF-H Domains of Twinfilin Play Functionally Distinct Roles in Interactions with Actin Monomers

Molecular Biology of the Cell ◽

10.1091/mbc.e02-03-0157 ◽

2002 ◽

Vol 13 (11) ◽

pp. 3811-3821 ◽

Cited By ~ 55

Author(s):

Pauli J. Ojala ◽

Ville O. Paavilainen ◽

Maria K. Vartiainen ◽

Roman Tuma ◽

Alan G. Weeds ◽

...

Keyword(s):

Binding Site ◽

Binding Activity ◽

Size Exclusion ◽

Actin Dynamics ◽

Actin Binding ◽

Actin Monomer ◽

Wild Type ◽

Native Page ◽

Exclusion Chromatography

Twinfilin is a ubiquitous and abundant actin monomer–binding protein that is composed of two ADF-H domains. To elucidate the role of twinfilin in actin dynamics, we examined the interactions of mouse twinfilin and its isolated ADF-H domains with G-actin. Wild-type twinfilin binds ADP-G-actin with higher affinity (K D = 0.05 μM) than ATP-G-actin (K D = 0.47 μM) under physiological ionic conditions and forms a relatively stable (k off = 1.8 s−1) complex with ADP-G-actin. Data from native PAGE and size exclusion chromatography coupled with light scattering suggest that twinfilin competes with ADF/cofilin for the high-affinity binding site on actin monomers, although at higher concentrations, twinfilin, cofilin, and actin may also form a ternary complex. By systematic deletion analysis, we show that the actin-binding activity is located entirely in the two ADF-H domains of twinfilin. Individually, these domains compete for the same binding site on actin, but the C-terminal ADF-H domain, which has >10-fold higher affinity for ADP-G-actin, is almost entirely responsible for the ability of twinfilin to increase the amount of monomeric actin in cosedimentation assays. Isolated ADF-H domains associate with ADP-G-actin with rapid second-order kinetics, whereas the association of wild-type twinfilin with G-actin exhibits kinetics consistent with a two-step binding process. These data suggest that the association with an actin monomer induces a first-order conformational change within the twinfilin molecule. On the basis of these results, we propose a kinetic model for the role of twinfilin in actin dynamics and its possible function in cells.

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Investigating the propagation kinetics of a novel class of nitrogen-containing methacrylates via PLP-SEC

Polymer Chemistry ◽

10.1039/c6py00867d ◽

2016 ◽

Vol 7 (26) ◽

pp. 4342-4351 ◽

Cited By ~ 11

Author(s):

Katrin B. Kockler ◽

Friederike Fleischhaker ◽

Christopher Barner-Kowollik

Keyword(s):

Size Exclusion Chromatography ◽

Rate Coefficient ◽

Pulsed Laser ◽

Propagation Rate ◽

Size Exclusion ◽

Arrhenius Parameters ◽

Exclusion Chromatography ◽

Kinetics Of ◽

Propagation Rate Coefficient ◽

Pulsed Laser Polymerization

The Mark–Houwink–Kuhn–Sakurada parameters as well as Arrhenius parameters of the propagation rate coefficient for a new group of nitrogen-containing methacrylates were determined via triple detector SEC and pulsed laser polymerization–size exclusion chromatography, respectively.

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New use of size exclusion chromatography in kinetics of mechanical degradation of polymers in solution

Journal of Applied Polymer Science ◽

10.1002/app.1993.070501201 ◽

1993 ◽

Vol 50 (12) ◽

pp. 2039-2045 ◽

Cited By ~ 20

Author(s):

Mircea Florea

Keyword(s):

Size Exclusion Chromatography ◽

Size Exclusion ◽

Mechanical Degradation ◽

Exclusion Chromatography ◽

Kinetics Of

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Kinetics of RAFT polymerization and copolymerization of vinyl monomers by size exclusion chromatography

European Polymer Journal ◽

10.1016/j.eurpolymj.2019.109356 ◽

2020 ◽

Vol 122 ◽

pp. 109356 ◽

Cited By ~ 4

Author(s):

Vaidas Klimkevicius ◽

Medeina Steponaviciute ◽

Ricardas Makuska

Keyword(s):

Size Exclusion Chromatography ◽

Raft Polymerization ◽

Size Exclusion ◽

Vinyl Monomers ◽

Exclusion Chromatography ◽

Kinetics Of

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Onchocerca retinol- and ivermectin-binding protein activity

Parasitology ◽

10.1017/s0031182000084791 ◽

1996 ◽

Vol 112 (2) ◽

pp. 221-225 ◽

Cited By ~ 8

Author(s):

P. G. Lal ◽

E. R. James

Keyword(s):

Adult Worm ◽

Specific Binding ◽

Binding Protein ◽

Binding Activity ◽

Retinol Binding Protein ◽

Size Exclusion ◽

Protein Activity ◽

Molecular Weights ◽

Molar Excess ◽

Exclusion Chromatography

SummaryThe presence of retinol-binding protein (RBP) activity in Onchocerca cervicalis adult worms and interaction with ivermectin has been studied using high pressure size exclusion chromatography (HPSEC). Four distinct peaks of [3H]-retinol incorporation were obtained corresponding to approximate molecular weights of 150, 67, 19·7 and 4–6 kDa, the 2 smaller Mr peaks accounting for most of the binding activity. Competition for binding using non-labelled retinol at 200-fold molar excess indicated that specific binding of retinol occurred only to the 19–7 kDa fraction. Competition by ivermectin also inhibited binding of [3H]-retinol to the third peak. Following incubation with [3H]-ivermectin & peaks of similar molecular weights were also detected by HPSEC in soluble adult worm homogenate, However, in this case the 150 kDa fraction was most prominent. Both non-labelled ivermectin and non-labelled retinol at 200-fold molar excess reduced binding of [3H]-ivermectin to all & fractions. These data indicate that the putative Onchocerca RBP has an approximate molecular weight of 19·7 kDa, that retinol also binds to 3 additional fractions non-specifically, that the pattern of binding of ivermectin to adult worm material is quantitatively and qualitatively different from the binding exhibited by retinol, and that ivermectin interferes with the binding of retinol to the 19·7 kDa Onchocerca protein.

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BuGZ facilitates loading of spindle assembly checkpoint proteins to kinetochores in early mitosis

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.013598 ◽

2020 ◽

Vol 295 (43) ◽

pp. 14666-14677

Author(s):

Hazheen K. Shirnekhi ◽

Jacob A. Herman ◽

Patrick J. Paddison ◽

Jennifer G. DeLuca

Keyword(s):

Spindle Assembly Checkpoint ◽

Spindle Assembly ◽

Size Exclusion ◽

Checkpoint Signaling ◽

Checkpoint Proteins ◽

Binding Partners ◽

Exclusion Chromatography ◽

Mitotic Spindle Assembly ◽

Kinetics Of ◽

Early Mitosis

BuGZ is a kinetochore component that binds to and stabilizes Bub3, a key player in mitotic spindle assembly checkpoint signaling. Bub3 is required for kinetochore recruitment of Bub1 and BubR1, two proteins that have essential and distinct roles in the checkpoint. Both Bub1 and BubR1 localize to kinetochores through interactions with Bub3, which are mediated through conserved GLEBS domains in both Bub1 and BubR1. BuGZ also has a GLEBS domain, which is required for its kinetochore localization as well, presumably mediated through Bub3 binding. Although much is understood about the requirements for Bub1 and BubR1 interaction with Bub3 and kinetochores, much less is known regarding BuGZ's requirements. Here, we used a series of mutants to demonstrate that BuGZ kinetochore localization requires only its core GLEBS domain, which is distinct from the requirements for both Bub1 and BubR1. Furthermore, we found that the kinetics of Bub1, BubR1, and BuGZ loading to kinetochores differ, with BuGZ localizing prior to BubR1 and Bub1. To better understand how complexes containing Bub3 and its binding partners are loaded to kinetochores, we carried out size-exclusion chromatography and analyzed Bub3-containing complexes from cells under different spindle assembly checkpoint signaling conditions. We found that prior to kinetochore formation, Bub3 is complexed with BuGZ but not Bub1 or BubR1. Our results point to a model in which BuGZ stabilizes Bub3 and promotes Bub3 loading onto kinetochores in early mitosis, which, in turn, facilitates Bub1 and BubR1 kinetochore recruitment and spindle assembly checkpoint signaling.

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