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Studies on the Recombinant Mutants of the Cys-298 Residue of Human Aldose Reductase

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Studies on the Recombinant Mutants of the Cys-298 Residue of Human Aldose Reductase Studies on the Recombinant Mutants of the Cys-298 Residue of Human Aldose Reductase by Emeka J. Udeigwe Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Science in the Chemistry Program YOUNGSTOWN STATE UNIVERSITY August, 2015 Studies on the Recombinant Mutants of the Cys-298 Residue of Human Aldose Reductase Emeka J. Udeigwe I hereby release this thesis to the public. I understand that this thesis will be made available from the OhioLINK ETD Center and the Maag Library Circulation Desk for public access. I also authorize the University or other individuals to make copies of this thesis as needed for scholarly research. Signature: _______________________________________________________________ Emeka J. Udeigwe, Student Date Approvals: _______________________________________________________________ Dr. Ganesaratnam K. Balendiran, Thesis Advisor Date _______________________________________________________________ Dr. Sherri Lovelace-Cameron, Committee Member Date _______________________________________________________________ Dr. Brian D. Leskiw, Committee Member Date _______________________________________________________________ Dr. Salvatore A. Sanders, Associate Dean of Graduate Studies Date © Emeka J. Udeigwe 2015 iii THESIS ABSTRACT The enzyme human aldose reductase (hAR) catalyzes the NADPH-dependent reduction of glucose to sorbitol. hAR is part of the polyol pathway and it exists in the native and the activated form. Of the three cysteine residues in hAR, located on the active site, the Cys- 298 residue is prone to oxidative modification. Previous studies have reported that the Cys-298-Ser mutant shows similar characteristics as the activated enzyme, and include an increased Vmax and Km, a comparatively low kcat/Km and a large Ki value. Using site- directed mutagenesis of Cys-298 to Asparagine and Glutamate (Cys-298-Asn and Cys- 298-Glu respectively), and DL-Glyceraldehyde as the substrate, the Cys-298-Asn mutant displayed a 60% increase in Km, a 65% decrease in kcat and an 18% increase in kcat/Km. The Cys-298-Glu mutant, however, displayed an increased Km, >3-fold, a 49% decrease in kcat, and a decreased kcat/km (up to 84%). The increased Km the Cys-298-Glu mutant enzyme demonstrates a reduced affinity for the substrate compared to the wild-type, suggesting a likelihood that the interaction of NADPH with the enzyme is weakened, hence a reduced conformational change of the enzyme-NADPH binary complex and a weaker substrate binding. It is possible that glutamic acid, an acidic, polar amino acid destabilizes the NADPH and the substrate-binding site. The increased Km and kcat of the Cys-298-Asn mutant shows an expected trend, due to the hydrophobic nature of asparagine and the ability of its carboxyamide group to form a hydrogen bond with neighboring residues on the active site pocket. The hydrogen bonding would permit the tight binding of NADPH to the enzyme, and the increased formation of the enzyme- NADPH binary complex. These results present facts about these mutants that have not been previously studied. iv ACKNOWLEDGEMENT My sincere gratitude goes to my advisor, Dr. Ganesaratnam Balendiran for the incredible opportunity to work in his research group, his support and guidance throughout the duration of this project and my studies, his consistent advice and his resilient spirit to ensure that I surpass my limitations. To Sudipti Gupta, for her enthusiastic and dauntless role in this research and for the indelible part she played in this project. My deepest appreciation also goes to the head, faculty and staff of the Chemistry Department for their support in ensuring the completion of my program. I would like to thank Dr. Sherri Lovelace-Cameron and Dr. Brian Leskiw for the privilege of being my committee members, especially to Dr. Sherri Lovelace-Cameron for her endless willingness to address my numerous concerns. To the members of my lab: Dan, DaVena, Heather, Amanda, Niloufar, Brian, Tamer and Nate. Working with this group of students has been nothing short of inspiring. To my good friend, Charles, for his encouragement when the going was tough; I could never ask for a better human being. I also need to thank most desperately my family, who has been the greatest support I have had my entire life. My brothers, Dr. Lawrence and Dr. Theo, for believing in me and giving me the support that I needed to become who I am today. To my mother, thank you for all your love and prayers, and to all my siblings for their constant support. Not to forget that this study would not have been possible without the immense contribution of Grant, DK085496, from the National Institutes of Health and the American Diabetes Association. v TABLE OF CONTENTS TITLE PAGE………………………………………………………………………… i SIGNATURE PAGE………………………………………..……………………….. ii COPYRIGHT PAGE…………………………………………...……………………. iii ABSTRACT…………………………………………………………………………. iv ACKNOWLEDGEMENTS….………………………………………………………. v TABLE OF CONTENTS……………………………………………………………. vi LIST OF TABLES…………………………………………………………………… x LIST OF FIGURES………………………………………………………………….. xii LIST OF SYMBOLS AND ABBREVIATIONS……………………………………. xv 1.0 INTRODUCTION…………………………………………………………….. 1 1.1 OVERVIEW OF RELATED TOPICS……………………………………. 2 1.1.1 The polyol pathway……………………………………………….. 2 1.1.2 Aldo-keto reductases……………………………………………… 4 1.1.3 Basic structural features of some aldo-keto reductases……………. 5 1.1.4 Aldose reductase…………………………………………………… 6 1.1.5 Variations, topology and structural features of aldose reductase…. 7 1.1.6 Catalytic mechanism of aldose reductase…………………………. 9 1.1.7 Native and activated forms of Aldose reductase…………………... 14 1.2 RELATED WORK………………………………………………………... 16 1.3 OBJECTIVE OF THESIS…………………………………………………. 35 vi 2.0 MATERIALS AND METHODS……………………………………………..... 37 2.1 Material……………………………………………………………….. 37 2.2 Buffers, strains and media……………………………………………. 38 2.2.1 pET 15b Vector………………………………………………. 40 2.2.2 pWhiteScript………………………………………………….. 42 2.2.3 pUC18……………………………………………………….... 42 2.3 METHODS…………………………………………………………… 44 2.3.1 Mutagenic primer design……………………………………... 44 2.3.2 Polymerase Chain Reaction……….……………………..…… 44 2.3.3 Dpn I Digestion of the amplification products……………….. 48 2.3.4 Preparation of competent cells by calcium chloride treatment. 49 2.3.5 Transformation of XL1-Blue supercompetent cells………….. 49 2.3.6 Plasmid preparation using QIAPrep spin miniprep kit……….. 50 2.3.7 DNA Sequencing…………………………………………….. 51 2.3.8 Transformation of BL21(DE3) competent E.coli cells………. 52 2.3.9 Sodium-dodecyl-sulfate polyacrylamide gel electrophoresis... 52 2.3.10 Protein expression……………………………………………. 54 vii 2.3.11 Sonication…………………………………………………….. 55 2.3.12 Immobilized metal affinity chromatography (TALON)….….. 56 2.3.13 Dialysis……………………………………………………….. 57 2.3.14 Protein quantification: Bradford reagent assay………………. 58 2.13.15 Thrombin cleavage of histidine-tag…………………………. 58 3.0 ENZYME KINETICS………………………………………………………… 59 3.1 Aldehyde reduction kinetics of the Cys-298-Asn and Cys-298-Glu mutants……………………………………………………………………. 59 3.2 Materials…………………………………………………………………... 59 3.3 Buffers, media and solutions……………………………………………… 59 3.4 Methods…………………………………………………………………… 60 4.0 RESULTS………………………………………………………………………. 62 4.1 DNA sequencing…………………………………………………………… 62 4.2 Transformation of BL21 (DE3) competent E.coli cells…………………… 75 4.3 Protein expression…………………………………………………………. 77 4.4 Immobilized metal affinity chromatography………………………………. 78 4.5 Protein dialysis…………………………………………………………….. 80 4.6 Protein quantification: Bradford reagent assay……………………………. 81 viii 4.7 Thrombin cleavage………………………………………………………… 82 4.8 Aldehyde reduction kinetics of the Cys-298-Asn and the Cys-298-Glu mutant enzyme, using DL -Glyceraldehyde as substrate…………………... 83 5.0 DISCUSSION………………………………………………………………….. 88 6.0 FUTURE WORK……………………………………………………………… 91 7.0 References……………………………………………………………………… 92 ix LIST OF TABLES 1.1 Kinetic parameters for native and oxidized aldose reductase determined using either recombinant wild-type human aldose reductase or aldose reductase isolated from human placenta [26]. As reported by Grimshaw et al., (1996)..….. 17 1.2 Kinetic parameters for wild type and mutant aldose reductase using different substrates [46]. As reported by Petrash et al., (1992)……………………………. 20 1.3 Kinetic parameters of reduced and carboxymethylated wild type and mutant aldose redcutase [46] As reported by Petrash et al., (1992)…………………………..... 22 1.3 Inhibitor sensitivity of the reduced and carboxymethylated aldose reductase [46]. As reported by Petrash et al., (1992)……………………………………………… 24 1.5 Comparison of the catalytic activity of the wild-type and the Cys-298-Ser mutants hAR for carbonyl reduction and alcohol oxidation reactions at 25C [1]……..... 29 2.1 Mutagenesis primers sequences, melting temperature and percentage GC content. 45 2.2 Mutagenesis sample reaction composition………………………………………. 46 2.3 Mutagenesis control reaction composition………………………………………. 47 2.4 Polymerase chain reaction cycling parameters for the QucikChange II Site- directed Mutagenesis………………………………………………….................. 48 2.5 Separating gel reagent compositions……………………………………………. 53 2.6 Stacking gel reagent compositions………………………………………………. 54 4.1 Rates of reaction and average change in absorbance per unit time for the Cys-298-Asn mutants, with different substrate concentrations of x DL -Glyceraldehyde…………..……………………………………...................... 84 4.2 Rates of reaction and average change in absorbance per unit time for the Cys-298-Glu mutants, with different substrate concentrations
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