Human deoxycytidine kinase (dCK) phosphorylates the natural deoxyribonucleosides deoxycytidine (dC), deoxyguanosine (dG) and deoxyadenosine (dA) and is an essential enzyme for the phosphorylation of numerous nucleoside analog prodrugs routinely used in cancer and antiviral chemotherapy. For many of these compounds, the phosphorylation step catalyzed by dCK is the rate-limiting step in their overall activation pathway. To determine the factors that limit the phosphorylation efficiency of the prodrug, we solved the crystal structure of dCK to a resolution of 1.6 A in complex with its physiological substrate deoxycytidine and with the prodrugs AraC and gemcitabine. The structures reveal the determinants of dCK substrate specificity. Especially relevant to new prodrug development is the interaction between Arg128 and the hydrogen-bond acceptor at the sugar 2'-arabinosyl position of AraC and gemcitabine. On the basis of the structures, we designed a catalytically superior dCK variant that could be used in suicide gene-therapy applications.

The rate of exchange of actin‐bound nucleotide is decreased by a factor of about 20 when actin is complexed with DNAase I without affecting the binding constant of calcium for actin. Binding constants of DNAase I to monomeric and filamentous actin were determined to be 5 × 108 M−1 and 1.2 × 104 M−1 respectively. The depolymerisation of F‐actin by DNAase I appears to be due to a shift in the G‐Fequilibrium of actin by DNAase I. Inhibition of the DNA‐degrading activity of DNAase I by G‐actin is of the partially competitive type.

Choline kinase, responsible for the phosphorylation of choline to phosphocholine as the first step of the CDP-choline pathway for the biosynthesis of phosphatidylcholine, has been recognized as a new target for anticancer therapy. Crystal structures of human choline kinase in its apo, ADP and phosphocholine-bound complexes, respectively, reveal the molecular details of the substrate binding sites. ATP binds in a cavity where residues from both the N and C-terminal lobes contribute to form a cleft, while the choline-binding site constitutes a deep hydrophobic groove in the C-terminal domain with a rim composed of negatively charged residues. Upon binding of choline, the enzyme undergoes conformational changes independently affecting the N-terminal domain and the ATP-binding loop. From this structural analysis and comparison with other kinases, and from mutagenesis data on the homologous Caenorhabditis elegans choline kinase, a model of the ternary ADP.phosphocholine complex was built that reveals the molecular basis for the phosphoryl transfer activity of this enzyme.

The observed changes of nucleotide state and conformation and the corresponding protein structural changes are correlated with intermediates occurring along the reaction coordinate and show the sequence of events occurring during phosphate transfer. The low catalytic activity of human TMPK appears to be determined by structural changes required to achieve catalytic competence and it is suggested that a mechanism might exist to accelerate the activity.

Guanylate kinase (GMPK) is a nucleoside monophosphate kinase that catalyzes the reversible phosphoryl transfer from ATP to GMP to yield ADP and GDP. In addition to phosphorylating GMP, antiviral prodrugs such as acyclovir, ganciclovir, and carbovir and anticancer prodrugs such as the thiopurines are dependent on GMPK for their activation. Hence, structural information on mammalian GMPK could play a role in the design of improved antiviral and antineoplastic agents. Here we present the structure of the mouse enzyme in an abortive complex with the nucleotides ADP and GMP, refined at 2.1 Å resolution with a final crystallographic R factor of 0.19 (R free ‫؍‬ 0.23). Guanylate kinase is a member of the nucleoside monophosphate (NMP) kinase family, a family of enzymes that despite having a low primary structure identity share a similar fold, which consists of three structurally distinct regions termed the CORE, LID, and NMP-binding regions. Previous studies on the yeast enzyme have shown that these parts move as rigid bodies upon substrate binding. It has been proposed that consecutive binding of substrates leads to "closing" of the active site bringing the NMP-binding and LID regions closer to each other and to the CORE region. Our structure, which is the first of any guanylate kinase with both substrates bound, supports this hypothesis. It also reveals the binding site of ATP and implicates arginines 44, 137, and 148 (in addition to the invariant P-loop lysine) as candidates for catalyzing the chemical step of the phosphoryl transfer.Guanylate kinase (GMPK, 1 ATP:GMP phosphotransferase, EC 2.7.4.8) is a critical enzyme for the biosynthesis of GTP and dGTP by catalyzing the phosphoryl transfer from ATP to (d)GMP resulting in ADP and (d)GDP (1, 2). GMPK also plays an important role in the recycling of the second messenger cGMP (3). In addition to these physiological roles, GMPK is essential for the activation of prodrugs used for the treatment of cancers and viral infections (4, 5). Therefore, it is medically important to elucidate its enzymatic mechanism and the structural basis for its nucleotide specificity. Our current structural understanding of this enzyme is derived from the apo-and GMP-bound structures of the yeast GMPK (6) and from analogy to other nucleoside monophosphate (NMP) kinases (7).It has been shown that the induced fit mechanism (8) plays an important role in NMP kinases, of which adenylate kinase is the best characterized (9 -11). NMP kinases catalyze phosphoryl transfer by binding both donor and acceptor nucleotides to form a ternary complex. Comparison of the crystal structures of nucleotide-free adenylate kinase to the one in which a single substrate is bound (AMP or ATP) and to the complex in which both substrates are present revealed the conformational changes that occur along the reaction coordinate: from an open unbound enzyme via a partially closed intermediate in which a single substrate is present to the fully closed form in the presence of both substrates. These substrate-induced conform...

The crystal structures of Escherichia coli thymidylate kinase (TmpK) in complex with P 1 -(5-adenosyl)-P 5 -(5-thymidyl)pentaphosphate and P 1 -(5-adenosyl)P 5 -[5-(3-azido-3-deoxythymidine)] pentaphosphate have been solved to 2.0-Å and 2.2-Å resolution, respectively. The overall structure of the bacterial TmpK is very similar to that of yeast TmpK. In contrast to the human and yeast TmpKs, which phosphorylate 3-azido-3-deoxythymidine 5-monophosphate (AZT-MP) at a 200-fold reduced turnover number (k cat ) in comparison to the physiological substrate dTMP, reduction of k cat is only 2-fold for the bacterial enzyme. The different kinetic properties toward AZT-MP between the eukaryotic TmpKs and E. coli TmpK can be rationalized by the different ways in which these enzymes stabilize the presumed transition state and the different manner in which a carboxylic acid side chain in the P loop interacts with the deoxyribose of the monophosphate. Yeast TmpK interacts with the 3-hydroxyl of dTMP through Asp-14 of the P loop in a bidentate manner: binding of AZT-MP results in a shift of the P loop to accommodate the larger substituent. In E. coli TmpK, the corresponding residue is Glu-12, and it interacts in a side-on fashion with the 3-hydroxyl of dTMP. This different mode of interaction between the P loop carboxylic acid with the 3 substituent of the monophosphate deoxyribose allows the accommodation of an azido group in the case of the E. coli enzyme without significant P loop movement. In addition, although the yeast enzyme uses Arg-15 (a glycine in E. coli) to stabilize the transition state, E. coli seems to use Arg-153 from a region termed Lid instead. Thus, the binding of AZT-MP to the yeast TmpK results in the shift of a catalytic residue, which is not the case for the bacterial kinase.Phosphorylation of AZT-MP by thymidylate kinase (TmpK; EC 2.7.4.9, ATP:dTMP phosphotransferase) has been implicated as the rate-limiting step in the activation pathway of the anti-HIV prodrug 3Ј-azido-3Ј-deoxythymidine (AZT). AZT must be phosphorylated by cellular enzymes to azidothymidine triphosphate before it can exert its antiviral effect by inhibiting HIV reverse transcriptase and acting as a chain terminator of nascent DNA strands. The bottleneck of AZT activation lies in the second phosphorylation step, the step that adds a phosphate group to azidothymidine monophosphate (AZT-MP) to yield azidothymidine diphosphate and is catalyzed by TmpK. Cells treated with AZT accumulate the toxic AZT-MP (1-3) in millimolar concentration (4, 5), and thus the active AZT triphosphorylated metabolite is at very low concentration. This situation is only slightly improved upon coinfection with herpes simplex virus I thymidine kinase-carrying vectors that efficiently phosphorylate the antiviral drugs acyclovir or ganciclovir but not AZT or AZT-MP (6, 7).Therefore, we set out to understand the structural basis for the slow activation of AZT-MP. The structures of the yeast TmpK (TmpK yeast ) complexed with either the physiological substrate d...

Nucleoside-based inhibitors of reverse transcriptase were the first drugs to be used in the chemotherapy of AIDS. After entering the cell, these substances are activated to their triphosphate form by cellular kinases, after which they are potent chain terminators for the growing viral DNA. The two main factors limiting their efficacy are probably interrelated. These are the insufficient degree of reduction of viral load at the commencement of treatment and the emergence of resistant variants of the virus. The reason for the relatively poor suppression of viral replication appears to be inefficient metabolic activation. Thus, for the most extensively used drug, 3'-azido-3'-deoxythymidine (AZT), whereas phosphorylation to the monophosphate is facile, the product is a very poor substrate for the next kinase in the cascade, thymidylate kinase. Because of this, although high concentrations of the monophosphate can be reached in the cell, the achievable concentration of the active triphosphate is several orders of magnitude lower. Determination of the structure of thymidylate kinase as a complex with AZT monophosphate (AZTMP) together with studies on the kinetics of its phosphorylation have now led to a detailed understanding of the reasons for and consequences of the poor substrate properties.

Asparaginases catalyze the hydrolysis of the amino acid asparagine to aspartate and ammonia. Bacterial asparaginases are used in cancer chemotherapy to deplete asparagine from the blood, since several hematological malignancies depend on extracellular asparagine for growth. To avoid the immune response against the bacterial enzymes it would be beneficial to replace them with human asparaginases. However, unlike the bacterial asparaginases, the human enzymes have a millimolar Km value for asparagine, making them inefficient in depleting the amino acid from blood. To facilitate the development of human variants suitable for therapeutic use, we solved the structure of human L-asparaginase (hASNase3). This asparaginase is an N-terminal nucleophile (Ntn) family member that requires autocleavage between Gly167 and Thr168 to become catalytically competent. For most Ntn-hydrolases this autoproteolytic activation occurs efficiently. In contrast, hASNas3 is relatively stable in its uncleaved state, and this allowed us to observe the structure of the enzyme prior to cleavage. To determine the structure of the cleaved state we exploited our discovery that the free amino acid glycine promotes complete cleavage of hASNase3. Both enzyme states were elucidated in the absence and presence of the product aspartate. Together, these structures provide insight into the conformational changes required for cleavage, and on the precise enzyme-substrate interactions. The new understanding of hASNase3 will serve to guide the design of variants that possess a decreased Km value for asparagine, making the human enzyme a suitable replacement for the bacterial asparaginases in cancer therapy.