Biochemical Characterization of Human Guanylate Kinase and Mitochondrial Thymidine Kinase: Essential Enzymes for the Metabolic Activation of Nucleoside Analog Prodrugs

Deoxyribonucleoside and nucleotide kinases are key enzymes that catalyze the critical phosphorylation steps in the conversion of antiviral and anticancer nucleoside analogs to their corresponding cytotoxic nucleoside triphosphates for incorporation into DNA. In this work, I characterized three enzymes: Human guanylate kinase (hGMPK), human mitochondrial thymidine kinase (hTK2), and E.coli guanosine-inosine kinase (ecGSK). They were recombinantly produced and kinetically characterized. A series of mutations were introduced to understand the catalytic roles of specific residues. The enzymes were structurally characterized for substrate-induced conformational changes, and two novel assays were devised to study their kinetics. Our main aim was to provide a basis for their potential use in cancer chemotherapy. The hGMPK was recombinantly produced in catalytically active form although it was previously reported to be inactive upon production in E.coli. In order to explain the role of certain residues in catalysis, a series of point mutations were introduced in hGMPK by rational design applying the structural information of mouse GMPK, which is 88% identical to hGMPK allowing us to build a homology model. Interestingly, it was found that a single hydrogen bond between the active site S37 and the carbonyl oxygen of guanine in GMP substrate is critical for binding of GMP and catalysis. Disturbing this single hydrogen bond in the form of the S37A mutation adversely affected the catalytic activity. Besides its catalytic role, S37 is required for the dynamics of the hinge part that connects two structural regions designated as NMP-binding region (NMP-BR) and the CORE region. Its mutation to proline (S37P), which is the least flexible amino acid in terms of sterically allowed conformations, reduced the catalytic efficiency of hGMPK by about 1000-fold making the molecule more like the non-enzymatic guanylate kinase domain of MAGUKs (membrane-associated guanylate kinase homologs). Similarly, the bidentate interaction of T83 with the carbonyl oxygen of guanine in GMP is required for catalysis. Y81 interacts with the phosphate of GMP and has a role in binary complex stabilization. We demonstrated in cell culture experiments that hGMPK, which catalyzes the second phosphorylation step in the final conversion of the antileukemic drug 6-thioguanine (6-TG) to 6-thioGTP/6-thiodGTP for incorporation into RNA and DNA, is the bottleneck enzyme in the metabolic activation of 6-TG, enhancing its cytotoxicity by several fold when overexpressed in HEK293 cells. Analyzing the SAXS structures of hGMPK in different conformational states, in particular in the open (unliganded) and completely closed (with two bound nucleotides) forms, revealed large conformational changes that occur during catalysis. The open-to-closed conformational transition of hGMPK induced by binding of ligands supports the model of the induced fit mechanism. In addition, we optimized the higher yield production of isotope-labeled (15N, 15N/13C) hGMPK for its structural analysis by NMR. GMP-induced 15N-1H HSQC (Heteronuclear Single Quantum Coherence) chemical shift changes for hGMPK mapped onto its open form confirmed our findings by SAXS studies that hGMPK undergoes substrate-induced conformational changes. In order to develop novel and advanced approaches for studying the catalytic properties of deoxyribonucleoside and nucleotide kinases, we devised two assays. In one assay, we used a CdS/ZnS quantum-dot (QDs)-modified gold electrode for the detection of hGMPK-catalyzed reaction in an enzyme-coupled assay based on the electrochemical sensing of NADH in a GMP concentration-dependent way. We also demonstrated the proof of concept of a light-controlled sensor for hGMPK immobilized on CdS/ZnS QDs-modified gold electrode. Similarly, we established an Amplex Red-based spectrophotometric and fluorometric enzyme-coupled assay for hGMPK as an alternative to the conventional NADH-dependent spectroscopic assay. Our new assay overcomes the overlapping wavelength problem associated with strong absorption of 6-thioguanine nucleotides at 340 nm, and it has the advantage of being usable both in the absorbance and fluorescence modes. We investigated the hGMPK loading capacity of calcium carbonate microparticles of different shapes. It was found that ellipsoidal microparticles with loaded hGMPK exhibited higher specific activities, after coating with polyelectrolytes, as compared to microparticles of all other shapes including spherical, rhomboidal, star and cube-like particles. Thus, ellipsoidal particles turned out to be more appropriate for drug loading and cellular targeting experiments. We determined the mitochondrial localization of hTK2 by expressing it in HEK293 cells as a fusion with C-terminal EGFP, and observed its subcellular localization by confocal microscopy. In addition, we solved the aggregation problem associated with hTK2 upon overexpression in E.coli. Our optimized protocol is based on the expression of hTK2 as a fusion with N-terminal His14-MBP-SUMObr tag (~60 kDa) under optimum conditions. Similarly, we generated a C- and N-terminal truncated form of hTK2 with improved catalytic activity as compared to wild-type hTK2, and optimized experimental conditions for its crystallization. Additionally, through directed evolution using error-prone PCR and subsequent screening of mutants against antiviral and anticancer nucleoside analogs, we found that two mutants designated M5 and M17 increased the sensitivity of the TK-deficient KY895 E.coli strain to gemcitabine by 25-fold and fourteen mutants by a factor of 10. Similarly, five mutants enhanced the sensitivity of KY895 to AZT by 3-fold. A unique property was explored for the recombinantly produced ecGSK that phosphorylates the nucleoside form of the clinically used antileukemic drug 6-thioguanine. To determine its structure-function relationship, experimental conditions were optimized for the crystallization of ecGSK.