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The fluorescence lifetimes and quantum efficiencies q of NADH, AcPyADH, and of the model compounds Ad-C3-NicH and Ad-Ce-NicH have been measured in water and in 1,2-propanediol solution in the temperature range 0-30°. For Ad-C3-NicH, NADH, and Ad-C6-NicH at 25°in water the absolute quantum efficiencies are 0.035, 0.019, and 0.017, respectively, to a precision of a few per cent, and the lifetimes are 0.70, 0.40, and 0.28 ± 0.03 to 0.05 nsec The contribution of the ground to lowest singlet transition to the absorption spectrum has been evaluated by fluorescence polarization observations, and from these and the molecular fluorescence spectra, emissive lifetimes Te have been calculated by the equation of Strickler and Berg. For NADH and AcPyADH under various conditions, the relation r = req is followed rigorously over a thirtyfold change in quantum efficiency. The absolute efficiency of quinine sulfate measured either by comparison with these derivatives or by the relation q = r/re turns out to be 0.70 ± 0.02 rather than the often quoted 0.55. The observed quantum efficiencies of energy transfer from the adenine to the dihydronicotinamide moiety were as follows for Ad-C3-NicH, NADH, and Ad-C6-NicH at 25°in aqueous solution: 0.44,0.34, and 0.10, respectively. For these three compounds, comparison of the rates and of the energies of activation for radiationless transitions calculated from the temperature dependence of the lifetimes shows that the quenching processes are essentially identical in 1,2-propanediol but different in aqueous solution, indicating that interactions in water, but not in 1,2-propanediol, are characteristic and specific for each compound. The simplified spectroscopic models, Ad-C3-NicH and Ad-Ce-NicH, which were designed to incorporate the absorption-emission chromophores of NADH by linking the adenyl and dihydronicotinamide moieties of NADH with triand hexamethylene chains, were prepared by dithionite reduction of the corresponding precursors possessing the nicotinamide ring in oxidized form. Accumulated spectroscopic evidence1*'3"18 indicates that coenzymes like NADH and FAD in aqueous solution tend to exist in "folded" or internally complexed forms (Figure 1) which resemble the "stacked" structures in the polynucleotides and nucleic acids. Because of the complicating features of the sugar and pyrophosphate moieties, full assessment of the factors that determine the folding can best be made by comparison with simpler models.19 This paper describes the results of parallel observations of the fluorescence properties of NADH, AcPyADH, and two synthetic spectroscopic model compounds, Ad-C3-NicH (5a) and Ad-C6-NicH (5b).2(1) (a) For the preceding paper on fluorescence lifetime studies, see

Aminoimidazole riobnucleotide carboxylase, the sixth step in the purine biosynthetic pathway, catalyzes the conversion of aminoimidazole ribonucleotide (AIR) to carboxyaminoimidazole ribonucleotide (CAIR). The gene products of the purE and purK genes (PurE and PurK, respectively) thought to be responsible for this activity have been overexpressed and the proteins purified to homogeneity. PurE separates from PurK in the first ammonium sulfate fractionation during the purification. No evidence for association of the two gene products under a variety of conditions using a variety of methods could be obtained. To facilitate the assay for CAIR production, the purC gene product, 5-aminoimidazole-4-N-succinylcarboxamide ribonucleotide (SAICAR) synthetase has also been overexpressed and purified to homogeneity. The activities of PurE, PurK, and PurE.PurK have been investigated. PurE alone is capable of catalyzing the conversion of AIR to CAIR 1 million times faster than the nonenzymatic rate. The Km for HCO3- in the PurE-dependent reaction is 110 mM! PurK possesses an ATPase activity that is dependent on the presence of AIR. No bicarbonate dependence on this reaction could be demonstrated (less than 100 microM), and AIR is not carboxylated during the hydrolysis of ATP. Incubation of a 1:1 mixture of PurE and PurK at low concentrations of bicarbonate (less than 100 microM) revealed that CAIR is produced but requires the stoichiometric conversion of ATP to ADP and Pi. No dependence on the concentration of HCO3- could be demonstrated. A new energy requirement in the purine biosynthetic pathway has been established.

We have shown that the reaction of guanosine with chloroacetaldehyde in aqueous solution in the physiological pH range yields 1,N2-ethenoguanosine (5,9-dihydro-9-oxo-3-~-~-ribofuranosylimidazo[l,2-a]purine). This compound could be hydrolyzed to 1,N2-ethenoguanine (5,9-dihydro-9-oxoimidazo[l,2-a]purine), which was also prepared authentically by hydriodic acid treatment of the glyoxal-guanine adduct. The 1,N2-ethenoguanine, which is an unsubstituted (at N-4, C-6, and C-7) Y-type base, is not fluorescent under the same conditions at which the 4-methyl compounds fluoresce. By contrast, the isomeric and angular N2,3-ethenoguanine (8,9-dihydro-9-oxoimidazo(2,l-blpurine) is fluorescent (bmitation 262 nm, Xemjmion 410 nm). The N2,3-ethenoguanine synthesis was initiated by the reaction of chloroacetaldehyde with 06-benzylguanine, 06-methylguanine, and 2-amino-6-benzylthiopurine, followed by hydrogenolysis or hydrolysis, hydrolysis, and oxidation and hydrolysis, respectively. The reaction of guanosine is indicative of the damage that can result from the action of the mutagen chloroacetaldehyde on guanosine derivatives under physiological pH conditions. The reaction of chloroacetaldehyde in aqueous solution with adenine-and cytosine-containing compounds1p2 to produce 1a6-and 3,N4-etheno-bridged compounds, respectively, has found wide a p p l i~a t i o n .~~~ Interest stems from the biological activity generally evident at the nucleoside, nucleotide, and coenzyme level and from the species responsible for the fluorescence emission properties.596 The crystal and molecular structures of suitable derivatives have been We agree with Kochetkov, Shibaev, and Kostl that in the pH range most favorable for reaction at 37 OC of chloroacetaldehyde with adenosine (pH 4.5) and cytidine (pH 3.5), guanosine is not reactive.2 When chloroacetaldehyde was used in this laboratory to modify tRNA in aqueous solution at different selected ~H S ,~ guanosine as well as cytidine and adenosine residues appeared to be undergoing attack at pH 6.3,lO within the optimum range for retention of tRNA tertiary structure.Since chloroacetaldehyde is known to be mutagenic12.13 and is one of the likely liver metabolites of vinyl chloride, its reaction with guanosine under physiological conditions was of particular interest. Moreover, the possible development of fluorescence due to the formation of an additional ring suggested the value of product comparison with the fluorescent natural nucleosides Y1p27 (wybutosine, Y -W Y O )~~ and YtZs3l (wyosine, W Y O )~~ and corresponding bases related to guanine.32The reaction of guanosine (1) with chloroacetaldehyde in aqueous solution at 37 "C was followed by the development of ultraviolet absorption at 305 nm over a period of hours and over a pH range from 6.5 to 4.5. At pH 6.5 the reaction rate is significant, but is still less than one-third that of adenosine with chloroacetaldehyde under the same conditions. The relative reaction rate for the guanosine reaction falls off sharply with decreasing pH and is practically ...

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We determined the dissociation constant (Kd) of a series of nucleotides for the bovine skeletal muscle type II catalytic subunit by displacing lin-benzoadenosine 5'-diphosphate (lin-benzo-ADP) with increasing concentrations of competing nucleotide. The Kd of each nucleotide was calculated from the decreases in the fluorescence polarization of lin-benzo-ADP that accompany its displacement from the catalytic subunit. We found that modifications of the adenine moiety reduce nucleotide affinity for the enzyme. The effect was most pronounced with modifications at position 6 of the base. Replacement of the 3'-hydroxyl group of ribose with a hydrogen increased the affinity of the nucleotide; addition of phosphate to the 2'- or 3'-hydroxyl groups, on the other hand, decreased nucleotide affinity. MgATP and MgADP exhibited Kd's of about 10 microM. AMP, which contains a negatively charged alpha-phosphate, bound with reduced affinity (643 microM). Adenosine, which lacks a charged alpha-phosphate, bound with a higher affinity (32 microM). To learn more about the nature of the alpha-phosphate binding site, a series of uncharged and positively charged derivatives of the 5'-position on the ribose moiety was prepared. The uncharged derivatives bound with much greater affinity than the negatively charged AMP. The Kd's for 5'-tosyladenosine and 5'-iodo-5'-deoxyadenosine were 30 and 32 microM, respectively. Like the negatively charged AMP, positively charged derivatives also bound less tenaciously than the neutral species. The positively charged 5'-amino-5'deoxyadenosine, for example, exhibited a 15-fold higher Kd (506 microM) than the neutral congenors.(ABSTRACT TRUNCATED AT 250 WORDS)

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