Anabel de Diego

Dehydroluciferyl-AMP is the main intermediate in the luciferin dependent synthesis of Ap4A catalyzed by firefly luciferase.

It was previously assumed that E·LH2‐AMP was the intermediate complex in the synthesis of Ap4A catalyzed by firefly luciferase (EC 1.13.12.7), when luciferin (LH2) was used as cofactor. However, here we show that LH2 is partly transformed, shortly after the onset of the luciferase reaction, to dehydroluciferin (L) with formation of an E·L‐AMP complex which is the main intermediate for the synthesis of Ap4A. Formation of three more derivatives of LH2 were also observed, related to the production of light by the enzyme. CoA, a known stimulator of light production, inhibits the synthesis of Ap4A by reacting with the E·L‐AMP complex and yielding L‐CoA.

Uridine 5′-polyphosphates (p4U and p5U) and uridine(5′)polyphospho(5′)nucleosides (UpnNs) can be synthesized by UTP:glucose-1-phosphate uridylyltransferase from Saccharomyces cerevisiae

UTP:glucose‐1‐phosphate uridylyltransferase (EC 2.7.7.9) from Saccharomyces cerevisiae can transfer the uridylyl moiety from UDP‐glucose onto tripolyphosphate (P3), tetrapolyphosphate (P4), nucleoside triphosphates (p3Ns) and nucleoside 5′‐polyphosphates (p4Ns) forming uridine 5′‐tetraphosphate (p4U), uridine 5′‐pentaphosphate (p5U) and dinucleotides, such as Ap4U, Cp4U, Gp4U, Up4U, Ap5U and Gp5U. Unlike UDP‐glucose, UDP‐galactose was not a UMP donor and ADP was not a UMP acceptor. This is the first example of an enzyme that may be responsible for accumulation of dinucleoside tetraphosphates containing two pyrimidine nucleosides in vivo. Occurrence of such dinucleotides in S. cerevisiae and Escherichia coli has been previously reported (Coste et al., J. Biol. Chem. 262 (1987) 12096–12103).

Synthesis of bisphosphonate derivatives of ATP by T4 RNA ligase

T4 RNA ligase catalyzes the synthesis of ATP β,γ‐bisphosphonate analogues, using the following substrates with the relative velocity rates indicated between brackets: methylenebisphosphonate (pCH2p) (100), clodronate (pCCl2p) (52), and etidronate (pC(OH)(CH3)p) (4). The presence of pyrophosphatase about doubled the rate of these syntheses. Pamidronate (pC(OH)(CH2–CH2–NH2)p), and alendronate (pC(OH)(CH2–CH2–CH2–NH2)p) were not substrates of the reaction. Clodronate displaced the AMP moiety of the complex E‐AMP in a concentration dependent manner. The K m values and the rate of synthesis (k cat) determined for the bisphosphonates as substrates of the reaction were, respectively: methylenebisphosphonate, 0.26 ± 0.05 mM (0.28 ± 0.05 s−1); clodronate, 0.54 ± 0.14 mM (0.29 ± 0.05 s−1); and etidronate, 4.3 ± 0.5 mM (0.028 ± 0.013 s−1). In the presence of GTP, and ATP or AppCCl2p the relative rate of synthesis of adenosine 5′,5‴‐P1,P4‐tetraphosphoguanosine (Ap4G) was around 100% and 33%, respectively; the methylenebisphosphonate derivative of ATP (AppCH2p) was a very poor substrate for the synthesis of Ap4G. To our knowledge this report describes, for the first time, the synthesis of ATP β,γ‐bisphosphonate analogues by an enzyme different to the classically considered aminoacyl‐tRNA synthetases.

Synthesis of (di)nucleoside polyphosphates by the ubiquitin activating enzyme E1

Previous work from this laboratory had shown that ligases may catalyze the synthesis of (di)nucleoside polyphosphates. Here, we show that one of the enzymes of the proteasome system (E1 or the ubiquitin (Ub) activating enzyme, EC 6.3.2.19) catalyzes very effectively (k cat = 0.29 ± 0.05 s−1) the transfer of AMP from the E–AMP–ubiquitin complex to tripolyphosphate or tetrapolyphosphate with formation of adenosine tetra‐ or pentaphosphate (p4A or p5A), respectively. Whereas the concomitant formation of AMP is stimulated by the presence of dithiothreitol in a concentration dependent manner, the synthesis of p4A is only slightly inhibited by this compound. Previous treatment of the enzyme (E1) with iodoacetamide inhibited only partially the synthesis of p4A. p4A can substitute for ATP as substrate of the reaction to generate the ubiquityl adenylate complex. A small amount of diadenosine pentaphosphate (Ap5A) was also synthesized in the presence of p4A.

Polyphosphates strongly inhibit the tRNA dependent synthesis of poly(A) catalyzed by poly(A) polymerase from Saccharomyces cerevisiae

Polyphosphates of different chain lengths (P3, P4, P15, P35), (1 μM) inhibited 10, 60, 90 and 100%, respectively, the primer (tRNA) dependent synthesis of poly(A) catalyzed poly(A) polymerase from Saccharomyces cerevisiae. The relative inhibition evoked by p4A and P4 (1 μM) was 40 and 60%, respectively, whereas 1 μM Ap4A was not inhibitory. P4 and P15 were assayed as inhibitors of the enzyme in the presence of (a) saturating tRNA and variable concentrations of ATP and (b) saturating ATP and variable concentrations of tRNA. In (a), P4 and P15 behaved as competitive inhibitors, with K i values of 0.5 μM and 0.2 μM, respectively. In addition, P4 (at 1 μM) and P15 (at 0.3 μM) changed the Hill coefficient (n H) from 1 (control) to about 1.3 and 1.6, respectively. In (b), the inhibition by P4 and P15 decreased V and modified only slightly the K m values of the enzyme towards tRNA.

Determination of enzymatic activities and metabolites in microliter sample size

A new generation of spectrophotometers able to measure a wide range of absorbance in microliter aliquots is currently used for the determination of DNA, RNA, and proteins. The object of this article is to show that these instruments could be easily adapted for routine evaluation of enzymes and metabolites in 1-2-microl volumes of biological samples.