https://www.cell.com/molecular-cell/pdf/S1097-2765(12)00696-X.pdf

Direct Regulation of GTP Homeostasis by (p)ppGpp: A Critical Component of Viability and Stress Resistance

Phosphorylation of nucleosides and nucleoside analogs by mammalian nucleoside monophosphate kinases

The catalytic mechanisms of adenylate kinase, guanylate kinase, uridylate kinase, and cytidylate kinase are reviewed in terms of kinetic and structural information that has been obtained in recent years. All four kinases share a highly related tertiary structure, characterized by a central five-stranded parallel beta-sheet with helices on both sides, as well as the three regions designated as the CORE, NMPbind, and LID domains. The catalytic mechanism continues to be refined to higher levels of resolution by iterative structure-function studies, and the strengths and limitations of site-directed mutagenesis are well illustrated in the case of adenylate kinase. The identity and roles of active site residues now appear to be resolved, and this review describes how specific site substitutions with unnatural amino acid side-chains have proven to be a major advance. Likewise, there is mounting evidence that phosphoryl transfer occurs by an associative transition state, based on (a) the stereochemical course of phosphoryl transfer, (b) geometric considerations, (c) examination of likely electronic distributions, (d) the orientation of the phosphoryl acceptor relative to the phosphoryl being transferred, (e) the most likely role of magnesium ion, (f) the lack of restricted access of solvent water, and (g) the results of oxygen-18 kinetic isotope. effect experiments.

Nucleoside monophosphate kinases: structure, mechanism, and substrate specificity.

Human deoxycytidine kinase activates the clinically established prodrugs AraC, fludarabine and cladribine and some recently developed chemotherapy agents such as gemcitabine and troxacitabine. In addition, dCK also phosphorylates the antiviral compounds 3TC and ddC used for the treatment of HIV infection. The critical function of dCK for the efficacy of prodrug chemotherapy is evident from a direct correlation between the activity of the enzyme and drug sensitivity in tumor cell lines 1 . Cells lacking dCK activity are resistant to a variety of drugs, including AraC, cladribine, fludarabine and gemcitabine 2,3 , and drug sensitivity to AraC can be restored by expressing functional dCK protein 4 . Moreover, in vivo studies conducted on animal tumors using gemcitabine showed that increased dCK activity, mediated by dCK gene transfer, results in enhanced accumulation and prolonged elimination kinetics of gemcitabine triphosphate and, ultimately, enhanced tumor response to the prodrug 5 . Despite the determining role played by human dCK in current anticancer prodrug treatment, no structural information on the enzyme was available until now. We have determined the three-dimensional structure of dCK to investigate the substrate specificity of the enzyme and to ultimately overcome the limitations imposed by the low catalytic activity of the enzyme: dCK is a relatively poor catalyst (with turnover rates well below 1 s ‐1 ). Our long-term goal is the structurebased design of mutant enzymes with enhanced activity to be used in gene therapy applications. Additionally, the protein structure may be used to guide the development of improved antiviral and anticancer molecules. We report the fully refined structures of human dCK in complex with dC‐ADP, AraC‐ADP‐Mg and gemcitabine‐ADP‐Mg. These crystal structures, with a natural nucleoside or nucleoside analogs and ADP‐Mg, represent ‘dead-end’ complexes composed of a mixture of substrates and products. Nonetheless, they allow us to observe a closed conformation of the enzyme similar to the one expected in the presence of ATP, as previously demonstrated in the case of other kinases (such as thymidylate kinase, TMPK) when comparing the ATP analog (β,γ-imidoadenosine 5′-triphosphate) to the ADP-bound structures 6 . Furthermore, analysis of distances in our dCK structures between the nucleosides and a modeled ATP molecule suggests that the position observed for the nucleosides in our ADP complex is similar to the one expected with ATP (see below). These structures explain the different phosphorylation efficiencies observed with various cytidine analogs and suggest specific modifications to our current arsenal of prodrugs that we predict will result in their improved phosphorylation by dCK and, thus, in an improved therapeutic index. Furthermore, the newly gained structural knowledge was exploited in the design of a dCK mutant that has a faster phosphorylation rate than the wild-type enzyme: ∼50-fold more efficient in phosphorylating deoxycytidine (dC) and ∼4-fold more efficient in activating gemcitabine. This engineered enzyme could have an important role in gene-directed enzyme prodrug therapy strategies. The presence of dCK variants with enhanced activity in neoplastic or virus-infected cells will confront the growing challenge of drug resistance 7 .

/pdf/structure-of-human-dck-suggests-strategies-to-improve-1zkmtzdhkh.pdf

Structure of human dCK suggests strategies to improve anticancer and antiviral therapy.

Intracellular metabolism and enzymatic phosphorylation of 9-(1,3-dihydroxy-2-propoxymethyl)guanine and acyclovir in herpes simplex virus-infected and uninfected cells.

Publisher Summary This chapter describes the purification procedure of guanylate kinases from human erythrocytes, hog brain, and rat liver. A convenient assay involves the measurement of both adenosine diphosphate (ADP) and guanosine diphosphate (GDP) formed from adenosine triphosphate (ATP) and guanosine monophosphate (GMP) to pyruvate kinase and lactate dehydrogenase reactions. The decrease in absorbancy at 340 nm associated with NADH oxidation is measured with a spectrophotometer using a blue filter. All procedures steps are performed at 0–4°. Enzymic activities and protein concentrations are determined after each step of purification. The purified guanylate kinases from the sources that are given in the chapter, were found stable for several years when stored below 0° in the presence of 70% saturated ammonium sulfate. In experiments with partially purified erythrocytic guanylate kinase, significant activity was retained for several minutes after precipitation at 3% perchloric acid followed by neutralization with potassium hydroxide.

R.E. Parks

Papers

Guanylate kinases from human erythrocytes, hog brain, and rat liver.