Structures of thymidine kinase 1 of human and mycoplasmic origin.

Cytosolic thymidine kinase 1, TK1, is a well known cell-cycle-regulated enzyme of importance in nucleotide metabolism as well as an activator of antiviral and anticancer drugs such as 3'-azido-3'-deoxythymidine (AZT). We have now determined the structures of the TK1 family, the human and Ureaplasma urealyticum enzymes, in complex with the feedback inhibitor dTTP. The TK1s have a tetrameric structure in which each subunit contains an alpha/beta-domain that is similar to ATPase domains of members of the RecA structural family and a domain containing a structural zinc. The zinc ion connects beta-structures at the root of a beta-ribbon that forms a stem that widens to a lasso-type loop. The thymidine of dTTP is hydrogen-bonded to main-chain atoms predominantly coming from the lasso loop. This binding is in contrast to other deoxyribonucleoside kinases where specific interactions occur with side chains. The TK1 structure differs fundamentally from the structures of the other deoxyribonucleoside kinases, indicating a different evolutionary origin.

Uropathogens and urinary tract concretion formation and catheter encrustations.

Infection stones (ammonium magnesium phosphate) and catheter encrustations have a common cause-urease producing microorganisms. With their rapid growth and frequent recurrences, infection stones are among the most troublesome of urinary system stones. For many patients with a long-term indwelling catheter, encrustations can be a severe problem. Urine composition is important, because, urine calcium enhances the crystallization process and urine citrate inhibits it. The role of non-urease producing microorganisms in stone forming processes is not well understood. Stones can now be successfully treated with a low morbidity index by percutaneous stone surgery or extracorporeal shock wave lithotripsy (ESWL) and recurrence of stone formation is then avoided by prolonged antibiotic treatment and oral citrate. Catheter encrustations and damage caused by ammonia released during urease activity can, however, be a serious problem in patients with indwelling catheters and our remedies are unsatisfactory.

Hydrolysis of urea by Ureaplasma urealyticum generates a transmembrane potential with resultant ATP synthesis.

When urea is added to Ureaplasma urealyticum, it is hydrolysed internally by a cytosolic urease. Under our measuring conditions, and at an external pH of 6.0, urea hydrolysis caused an ammonia chemical potential equivalent to almost 80 mV and, simultaneously, an increase in proton electrochemical potential (delta p) of about 24 mV with resultant de novo ATP synthesis. Inhibition of the urease with the potent inhibitor flurofamide abolished both the chemical potential and the increase of delta p such that ATP synthesis was reduced to approximately 5% of normally obtained levels. Uncouplers of electrochemical gradients had little or no effect on these systems. The electrochemical parameters and ATP synthesis were measured similarly at three other external pH values. Any change in delta p was primarily via membrane potential (delta psi), and the level of de novo ATP synthesis was related to the increase in delta p generated upon addition of urea and more closely to the ammonia chemical potential. Although the organisms lack an effective mechanism for internal pH homeostasis, they maintained a constant delta pH. The data reported are consistent with, and give evidence for, the direct involvement of a chemiosmotic mechanism in the generation of around 95% of the ATP by this organism. Furthermore, the data suggest that the ATP-generating system is coupled to urea hydrolysis by the cytosolic urease via an ammonia chemical potential.