Zhi-Rong Lü

The Effect of Histidine Residue Modification on Tyrosinase Activity and Conformation: Inhibition Kinetics and Computational Prediction

Abstract We found that the histidine chemical modification of tyrosinase conspicuously inactivated enzyme activity. The substrate reactions with diethylpyridinecarbamate showed slow-binding inhibition kinetics (K I = 0.24 ± 0.03 mM). Bromoacetate, as another histidine modifier, was also applied in order to study inhibition kinetics. The bromoacetate directly induced the exposures of hydrophobic surfaces following by complete inactivation via ligand binding. For further insights, we predicted the 3D structure of tyrosinase and simulated the docking between tyrosinase and diethylpyridinecarbamate. The docking simulation was shown to the significant binding energy scores (-3.77 kcal/mol by AutoDock4 and −25.26 kcal/mol by Dock6). The computational prediction was informative to elucidate the role of free histidine residues at the active site, which are related to substrate accessibility during tyrosinase catalysis.

The effect of rutin on arginine kinase: inhibition kinetics and thermodynamics merging with docking simulation.

Arginine kinase (AK; EC 2.7.3.3) is a key enzyme in the cellular energy metabolism of insects. Screening on potential effective inhibitors of AK may provide a pathway for novel, environmentally friendly insecticides. The results in this study indicated that rutin, as a noncompetitive inhibitor, interacts with AK mainly by a hydrophobic force forming an intermolecular complex with AK, which is according to the thermodynamic parameters obtained. Using a flexible docking method (AutoDock) the interaction between rutin and AK were further analyzed, which suggested in order to screen effective inhibitors, flexible active sites of AK (Ser63, Gly64, Val65, Tyr68) should be taken in account.

Towards Profiling the Gene Expression of Tyrosinase-induced Melanogenesis in HEK293 Cells: a Functional DNA Chip Microarray and Interactomics Studies

Abstract The overexpression of a single tyrosinase gene can induce conspicuous pigmentation in nonpigmented cells. We hypothesized that some unknown tyrosinase-associated genes are simultaneously regulated by melanin synthesis. To improve understanding of melanogenesis and tyrosinase-associated functions, we attempted to profile the genes that are altered during melanin production in HEK293 cells by using a functional DNA chip microarray. The candidate genes were obtained based on significance analysis of microarray (SAM) and further computational prediction via protein-protein interaction (PPI) mapping suggested that newly detected hub genes were involved in melanogenesis. PPI mapping using bioinformatic tools revealed 8 genes that formed an interaction hub. The yeast two-hybridization results suggested some candidate genes could interact with tyrosinase. The present study provides information to further understand the complex factors associated with tyrosinase- induced melanogenesis and apoptosis. The approach of combining expression data analysis and predicted protein interaction partners can help identify genes involved in pigmentation.

Alpha-Glucosidase Folding During Urea Denaturation: Enzyme Kinetics and Computational Prediction

In this study, we investigated structural changes in alpha-glucosidase during urea denaturation. Alpha-glucosidase was inactivated by urea in a dose-dependent manner. The inactivation was a first-order reaction with a monophase process. Urea inhibited alpha-glucosidase in a mixed-type reaction. We found that an increase in the hydrophobic surface of this enzyme induced by urea resulted in aggregation caused by unstable folding intermediates. We also simulated the docking between alpha-glucosidase and urea. The docking simulation suggested that several residues, namely THR9, TRP14, LYS15, THR287, ALA289, ASP338, SER339, and TRP340, interact with urea. Our study provides insights into the alpha-glucosidase unfolding pathway and 3D structure of alpha-glucosidase.

Kinetics of Zn2+-induced Brain Type Creatine Kinase Unfolding and Aggregation

We studied the effect of Zn2+ on the folding and aggregation of brain creatine kinase (CK-BB). We developed a method to purify CK-BB from rabbit brain and conducted inhibition kinetics and unfolding studies of CK-BB. Zn2+ conspicuously aggregated and osmolytes, such as glycine and proline, were able to suppress the formation of aggregates and protect the enzymatic activity against Zn2+. These results suggest that Zn2+ might act as a risk factor for CK-BB in the brain under certain conditions, and some osmolytes may help CK-BB to sustain the active state when Zn2+ is present. Our study provides useful information regarding the effect of Zn2+ on brain-derived metabolic enzymes, especially those that are putatively related to brain disease. Furthermore, our study suggests that although Zn2+ may induce CK-BB inactivation and misfolding, the ability of some abundant proteins and osmolytes to chelate Zn2+ nonspecifically may protect CK-BB and allow it to exist in the active form.

The Effect of Zn2+ on Human Brain Creatine Kinase: Unfolding and Aggregation Studies

Abstract We studied the effects of Zn2+ on human brain creatine kinase (HBCK). Zn2+ inactivated the activity of HBCK in a dose dependent manner (IC50 = 0.06 mM). The time-interval kinetic studies showed that the inactivation followed first-order reaction kinetics with a biphasic process. The spectroflurorimetry results showed that Zn2+ conspicuously induced the tertiary structural change of HBCK with exposure of its hydrophobic surfaces. On the contrary, the secondary structure was slightly changed by Zn2+. We also found that HBCK aggregation was induced by Zn2+. This aggregation was dependent on the temperature and the enzyme and Zn2+ concentrations. Some added osmolytes such as glycine and proline were able to successfully block CK aggregation and fully recover the conformation and activity of HBCK. Our study provides important insight into the unfavorable effect of Zn2+ on HBCK and it increases the understanding of the Zn2+ ligand-binding mechanism to the metabolic brain enzyme.

The effects of acrylamide on brain creatine kinase: Inhibition kinetics and computational docking simulation

The occurrence of acrylamide is frequently observed in processed foods. Therefore, the harmful effects of acrylamide on metabolic enzymes are important to understand. We studied the inhibitory effects of acrylamide on the brain creatine kinase (CK-BB). We found that CK-BB was kinetically inactivated by acrylamide accompanied by the disruption of the hydrophobic surface. Acrylamide mainly interacted with the thiol (-SH) residue of CK-BB and resulted in alkylation. A computational docking simulation supported that acrylamide directly bound to the active site of CK-BB where cysteine and glycine residues interacted mainly. The inhibition kinetics combined with computational prediction can be useful in order to have insights into the mechanisms regarding environmentally hazardous factors at the molecular level.

Effect of Cysteine Modification on Creatine Kinase Aggregation

We studied the effect of cysteine modification on creatine kinase (CK) aggregation as well as the kinetics of the process. We found that CK aggregation was modulated by different pH conditions in the presence of Zn2+, which is a CK aggregation trigger. The CK aggregation followed first-order kinetics, and this was effectively suppressed in acidic conditions. Even under the acidic condition, cysteine modification at the active site with using 5,5′-dithiobis-2-nitrobenzoic acid (DTNB) induced conspicuous aggregation in a dose-dependent manner. This aggregation process is directly related with decreasing the change of transition free-energy (ΔΔGAG). When dithiothreitol (DTT) was applied to the reaction system, the aggregates were significantly reduced: DTT treatment can fully reactivate (higher than 80%) the inactive CK that was separated from CK aggregates, whereas CK was completely inactivated by Zn2+ and DTNB. Some added osmolytes such as glycine and proline were able to successfully block CK aggregation by increasing the ΔΔGAG as well as by suppressing the hydrophobic CK surface. Our study suggests the effect of cysteine modification on the unfavorable aggregation of CK and on the aggregation process that followed first-order kinetics with the accompanying changes of transitional free energy and disruptions of the hydrophobic surface. We also demonstrate the successful protocol to block the aggregation.

Activation and Inactivation of Horseradish Peroxidase by Cobalt Ions

Abstract The effect of cobalt ions (Co2+) on horseradish peroxidase (HRP) was studied in vitro by enzymatic activity assay, electronic absorption spectra, intrinsic fluorescence spectra and 8-anilo-1-naphthalenesulfonate(ANS)-binding fluorescence spectra. Co2+ at concentrations below 0.1 mM mildly increased the HRP activity, whereas higher concentrations of Co2+ significantly inactivated HRP in a time and concentration-dependent manner. Steady-state kinetic studies show that Co2+ was a noncompetitive inhibitor of o-dianisidine oxidation by HRP. The Ki value dropped as the incubation time increased. Furthermore, Co2+ was found to be an uncompetitive inhibitor of H2O2. These results suggested that Co2+ would slowly bind to the enzyme and progressively induce conformational changes. Spectroscopic analysis showed that even for high Co2+ concentrations, the structure of HRP as a whole only changed slightly; however, there were significant conformational changes near or in the active site of HRP. Based on the above results, we suggest that Co2+ may bind with some amino acids near or in the active site of HRP and the conformational changes of HRP induced by such binding should be the main reason for activation and inactivation effect of Co2+. The potential binding sites of Co2+ were also proposed.

The Effect of Ag+ on Arginine Kinase: Inhibition Kinetics

Abstract We studied the effects of Ag+ on arginine kinase (AK) from Fenneropenaeus chinensis. Ag+ inactivated the activity of AK in a dose dependent manner (IC 50 = 15 μM). Kinetic studies showed that the inactivation of AK by Ag+ was reversible and occurred in a noncompetitive inhibition manner (K i = 2.8 μM). Spectroflurorimetry results showed that Ag+ did not induce conspicuous tertiary structural changes in AK at the corresponding concentration ranges of inactivation studies. However, the secondary structure measured by circular dichroism was slightly changed by Ag+. Taken together, these data suggest that the active site of AK is flexible, with the complete loss of activity occurring prior to significant changes in overall structures. Our study provides important insight into the inhibitory mechanism of Ag+ on AK and increases our understanding of the influence of Ag+ on the mechanism of this metabolic enzyme.