High-affinity ATP Site Research Articles

Kinetic characterization of Na,K-ATPase inhibition by Eosin

Eosin is a probe for the Na pump nucleotide site. In contrast to previous studies examining eosin effects on Na only ATPase, we examined Na,K-ATPase- and K-activated pNPPase activity in red blood cell membranes and purified renal Na,K-ATPase. At saturating ATP (3 mM) the eosin IC 50 for Na pump inhibition was 19 μM. Increasing ATP concentrations (0.2–2.5 mM) did not overcome eosin-induced inhibition, thus eosin is a mixed-type inhibitor of ATPase activity. To test if eosin can bind to the high-affinity ATP site, purified Na,K-ATPase was labeled with 20 μM FITC. With increasing eosin concentrations (0.1 μM–10 μM) the incorporation of FITC into the ATP site significantly decreases suggesting that eosin prevents FITC reaction at the high-affinity ATP site. Eosin was a more potent inhibitor of K-activated phosphatase activity than of Na,K-ATPase activity. At 5 mM pNPP the eosin IC 50 for Na pump inhibition was 3.8 ± 0.23 μM. Increasing pNPP concentrations (0.45–14.5 mM) did not overcome eosin-induced inhibition, thus eosin is a mixed-type inhibitor of pNPPase activity. These results can be fit by a model in which eosin and ATP bind only to the nucleotide site; in some pump conformations, this site is rigid and the binding is mutually exclusive and in other conformations, the site is flexible and able to accommodate both eosin and ATP (or pNPP). Interestingly, eosin inhibition of pNPPase became competitive after the addition of C 12E 8 (0.1%) but the inhibition of ATPase remained mixed.

TNP-8N3-ADP Photoaffinity Labeling of Two Na,K-ATPase Sequences under Separate Na+ plus K+ Control

ATP has high- and low-affinity effects on the sodium pump and other P-type ATPases. We have approached this question by using 2',3'-O-(trinitrophenyl)-8-azidoadenosine 5'-diphosphate (TNP-8N(3)-ADP) to photoinactivate and label Na,K-ATPase, both in its native state and after covalent FITC block of its high-affinity ATP site. With the native enzyme, the photoinactivation rate constant increases hyperbolically with a K(D(TNP-8N)3(-)(ADP)) of 0.11 microM; TNP-ATP and ATP protect the site with high affinities. The inactivation does not require Na(+), but K(+) inhibits with a K(K)' of 12 microM; Na(+) reverses this effect, with a K(Na) of 0.17 mM. This pattern suggests that Na(+) and K(+) are binding at sites in their "intracellular" conformation. It was known that FITC did not abolish the reverse phosphorylation by P(i), or the K(+)-phosphatase activity, and that TNP-8N(3)-ADP could subsequently photoinactivate the latter with >100-fold lower affinity; in that case, the cation sites acted as if facing outward [Ward, D. G., and Cavieres, J. D. (1998) J. Biol. Chem. 273, 14277-14284, 33759-33765]. Native and FITC-modified enzymes have now been photolabeled with TNP-8N(3)-[alpha-(32)P]ADP and alpha-chain soluble tryptic peptides separated by reverse-phase HPLC. With native Na,K-ATPase, three labeled peaks lead to the unique sequence alpha-(470)Ile-Val-Glu-Ile-Pro-Phe-Asn-Ser-Thr-Asn-X-Tyr-Gln-Leu-Ser-Ile-His-Lys(487), the dropped residue being alphaLys480. With the FITC enzyme, instead, two independent labeling and purification cycles return the sequence alpha-(721)Ala-Asp-Ile-Gly-Val-Ala-Met-Gly-Ile-Ala-Gly-Ser-Asp-Val-Ser-Lys(736). These results suggest that Na,K-ATPase also has a low-affinity nucleotide binding region, one that is under distinctive allosteric control by Na(+) and K(+). Moreover, the cation effects seem compatible with a slow, passive Na(+)/K(+) carrier behavior of the FITC-modified sodium pump.

Replacement of Several Single Amino Acid Side Chains Exposed to the Inside of the ATP-binding Pocket Induces Different Extents of Affinity Change in the High and Low Affinity ATP-binding Sites of Rat Na/K-ATPase

To investigate the relationship between the high and the low affinity ATP-binding site, which appears during the Na(+)/K(+)-ATPase reaction, four amino acids were mutated, the side chains of which are exposed to inside of the ATP-binding pocket. Six mutants, F475Y, K480A, K480E, K501A, K501E, and R544A, where the numbers correspond to the pig Na(+)/K(+)-ATPase alpha-chain, were expressed in HeLa cells. The apparent affinities were determined by high affinity ATP-dependent phosphorylation and by the low affinity activation of Na(+)/K(+)-ATPase or low affinity ATP inhibition of K(+)-para-nitrophenylphosphatase (pNPPase). For the mutants K480A and K501A, little affinity change was detected for either the high affinity or the low affinity effect. In contrast, the other four mutants reduced both apparent affinities. Strikingly, R544A had a 30-fold greater effect on the high affinity ATP site than the low affinity site. For the F475Y mutant, it is likely that there was a greater effect on the low affinity site than the high affinity site, but for both F475Y and K480E the affinity for the low affinity ATP effect was reduced so much that it was not possible to estimate a K(0.5). However, both the affinities for the K480E were reduced to approximately 1/20. The turnover number of the Na(+)/K(+)-ATPase and the apparent affinity for Na(+) and pNPP was reduced slightly or not at all for these mutants, but the turnover number of K(+)-pNPPase and the apparent affinity for K(+) were increased. These and other data suggest the presence of only one ATP-binding site, which can change its conformation to accept ATP with a high and low affinity. The requirement of Arg-544 and possibly Lys-501 is more important in forming a high affinity ATP binding conformation, and Phe-475 and possibly Lys-480 are more important in forming the low affinity ATP binding conformation.

Ligands Presumed to Label High Affinity and Low Affinity ATP Binding Sites Do Not Interact in an (αβ)2 Diprotomer in Duck Nasal Gland Na+,K+-ATPase, nor Do the Sites Coexist in Native Enzyme

The interaction of ligands deemed to be ATP analogues with renal Na(+),K(+)-ATPase suggests that two ATP binding sites coexist on each functional unit. Previous studies in which fluorescein 5-isothiocyanate (FITC) was used to label the high affinity ATP site and 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-diphosphate (TNP-ADP) was used to probe the low affinity site suggested that the two sites coexist on the same alphabeta protomer. Other studies in which FITC labeled the high affinity site and erythrosin-5-isothiocyanate (ErITC) labeled the low affinity site led to the conclusion that the high and low affinity sites exist on separate interacting protomers in a functional diprotomer. We report here that at 100% inhibition of ATPase activity by FITC, each alphabeta protomer of duck nasal gland enzyme has a single bound FITC. Both TNP-ADP and ErITC interact with FITC-bound protomers, which unambiguously demonstrates that putative high and low affinity ATP sites coexist on the same protomer. In unlabeled nasal gland enzyme, TNP-ADP and ErITC inhibit both ATPase activity and p-nitrophenyl phosphatase activity, functions attributed to the putative high and low affinity ATP site, respectively, by interacting with a single site with characteristics of the high affinity ATP binding site. In FITC-labeled enzyme, TNP-ADP and ErITC inhibit p- nitrophenyl phosphatase activity but at much higher concentrations than with the unmodified enzyme. Low affinity sites do not exist on the unmodified enzyme but can be detected only after the high affinity site is modified by FITC.

Photoinactivation of Fluorescein Isothiocyanate-modified Na,K-ATPase by 2′(3′)-O-(2,4,6-Trinitrophenyl)8-azidoadenosine 5′-Diphosphate: ABOLITION OF E1 AND E2 PARTIAL REACTIONS BY SEQUENTIAL BLOCK OF HIGH AND LOW AFFINITY NUCLEOTIDE SITES

The Na,K-ATPase activity of the sodium pump exhibits apparent multisite kinetics toward ATP, a feature that is inherent to the minimal enzyme unit, the alpha beta protomer. We have argued that this should arise from separate catalytic and noncatalytic sites on the alpha beta protomer as fluorescein isothiocyanate (FITC) blocks a high affinity ATP site on all alpha subunits and yet the modified Na, K-ATPase retains a low affinity response to nucleotides (Ward, D. G., and Cavieres, J. D. (1996) J. Biol. Chem. 271, 12317-12321). We now find that 2'(3')-O-(2,4,6-trinitrophenyl)8-azido-adenosine 5'-diphosphate (TNP-8N3-ADP), a high affinity photoactivatable analogue of ATP, can inhibit the K+-phosphatase activity of the FITC-modified enzyme during assays in dimmed light. The inhibition occurs with a Ki of 140 microM at 20 mM K+; it requires the adenine ring as 2'(3')-O-(2,4 6-trinitrophenyl) (TNP)-UDP or TNP-uridine are less potent and 2,4,6-trinitrobenzene-sulfonate is ineffective. Under irradiation with UV light, TNP-8N3-ADP inactivates the K+-phosphatase activity of the fluorescein-enzyme and also its phosphorylation by [32P]Pi. The photoinactivation process is stimulated by Na+ or Mg2+, and is inhibited by K+ or excess TNP-ADP. In the presence of 50 mM Na+ and 1 mM Mg2+, TNP-8N3-ADP photoinactivates with a K0.5 of 15 microM. Furthermore, TNP-8N3-ADP photoinactivates the FITC-modified, solubilized alpha beta protomers, even more effectively than the membrane-bound fluorescein-enzyme. These results strongly suggest that catalytic and allosteric ATP sites coexist on the alpha beta protomer of Na,K-ATPase.

Nucleotide Binding to the C-terminal Nucleotide Binding Domain of ArsA: STUDIES WITH AN ATP ANALOGUE, 5′-p-FLUOROSULFONYLBENZOYLADENOSINE (FSBA)

ArsA protein, the catalytic component of the plasmid-encoded anion-translocating ATPase in Escherichia coli, contains two consensus nucleotide binding domains, A1 and A2, that are connected by a flexible linker. ATP has previously been shown to cross-link to the A1 domain upon activation with UV light but not to the A2 domain. The ATP analogue, 5'-p-fluorosulfonylbenzoyladenosine (FSBA) was used to probe the nucleotide binding domains of ArsA. The covalently labeled protein was subjected to partial trypsin proteolysis, followed by Western blot analysis of the fragments with the anti-FSBA serum. The N-terminal amino acid sequence of the labeled fragment showed that FSBA binds preferentially to the C-terminal domain A2 both in the absence and the presence of antimonite. Occupancy of the two nucleotide binding sites was determined by protection from trypsin proteolysis. Trypsin cleaved the ArsA protein at Arg290 in the linker to generate a 32-kDa N-terminal and a 27-kDa C-terminal fragment. The 32-kDa fragment is compact and largely inaccessible to trypsin; however, the 27-kDa was cleaved further. Incubation with FSBA, which binds to the C-terminal domain, resulted in significant protection of the 27-kDa fragment. This fragment was not protected upon incubation with ATP alone, indicating that A2 might be unoccupied. However, upon incubation with ATP and antimonite, almost complete protection from trypsin was seen. ATP and FSBA together mimicked the effect of ATP and antimonite, implying that this fully protected conformation might be the result of both sites occupied with the nucleotide. It is proposed that the A1 site in ArsA is a high affinity ATP site, whereas the allosteric ligand antimonite is required to allow ATP binding to A2, resulting in catalytic cooperativity. Thus antimonite binding may act as a switch in regulating ATP binding to A2 and hence the ATPase activity of ArsA.

Binding of 2′(3′)-O-(2,4,6-Trinitrophenyl)ADP to Soluble αβ Protomers of Na,K-ATPase Modified with Fluorescein Isothiocyanate: EVIDENCE FOR TWO DISTINCT NUCLEOTIDE SITES

The overall reaction of well-defined solubilized protomers of Na,K-ATPase (one alpha plus one beta subunit) retains the dual ATP dependence observed with the membrane-bound enzyme, with distinctive ATP effects in the submicromolar and submillimolar ranges (Ward, D. G., and Cavieres, J. D. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 5332-5336). We have now found that the K+/-phosphatase activity of the alpha beta protomers is still inhibited by 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-diphosphate (TNP-ADP). What is most significant is that the TNP-ADP effect can be observed clearly with protomeric enzyme whose high affinity ATP site has been blocked covalently with fluorescein isothiocyanate. We conclude that nucleotides can bind at two discrete sites in each protomeric unit of Na,K-ATPase.

Na(+)-ATPase activity of Na(+),K(+)-ATPase. Reactivity of the E2 form during Na(+)-ATPase turnover.

Based on work of Post et al. (Post, R. L. Toda, G., and Rogers, F.N. (1975) J. Biol, Chem. 250, 691-701), we studied the E2 form reactivity of Na(+),K(+)-ATPase (EC 3.6.1.37) during Na(+)-ATPase turnover by following ATP hydrolysis with and without P(i) and enzyme phosphorylation from P(i) at 20 degrees C. For theoretical calculations we employed the Albers-Post scheme assuming that, even with no K+, E2 exhibits the ATP regulatory site. Using available rate constants the model predicts: (i) without P(i), Na(+)-ATPase displays a single high affinity ATP site but becomes double Michaelian (with high and low ATP affinity) when P(i) is present. (ii) Phosphorylation from P(i) can be detected during Na(+)-ATPase (t 1/2 about 400 ms); the KmP(i) is substantially higher that the KdP(i). (iii) P(i) incorporation is reduced by ATP acting with low affinity; this does not require an increase in the E2-E1 transition rate. (iv) The KmATP of the regulatory site is augmented when [P(i)] increases. The experimental observations, using partially purified pig kidney enzyme, agreed with the predictions. In addition they showed that: (i) extracellular Na+ can prevent P(i) incorporation; this effect is additive with that of ATP but with independent Ki values. (ii) Mg2+ stimulates P(i) incorporation with low affinity (Km of 1.5 mM). (iii) beta, gamma-Methyleneadenosine 5'-triphosphate and palmitoyl-CoA antagonize P(i) inhibition of Na(+)-ATPase. These results agree with a model where the Na(+),K(+)-ATPase and Na(+)-ATPase cycles share most of their intermediate steps and enzyme conformations.

Modification of the E1ATP binding site of Na+/K(+)-ATPase by the chromium complex of adenosine 5'-[beta,gamma-methylene]triphosphate blocks the overall reaction but not the partial activities of the E2 conformation.

The chromium complex of adenosine 5'-[beta,gamma-methylene]triphosphate, Cr(H2O)4AdoPP[CH2]P, inactivates Na+/K(+)-ATPase from pig kidney at 37 degrees C with an inactivation velocity constant of 7.1 x 10(-3) min-1 by binding to the high-affinity ATP site (E1ATP site). The dissociation constant (Kd) of the analogue at this site is 26 microM, and of ATP 0.8 microM. Inactivation of the overall reaction of Na+/K(+)-ATPase by Cr(H2O)4AdoPP[CH2]P did not alter the activities of the E2 conformational state such as K(+)-activated p-nitrophenylphosphatase, 86Rb+ occlusion and [3H]ouabain binding by the 'backdoor' phosphorylation. However, [3H]ouabain binding via the forwards reaction from E1ATP in the presence of Na+ + Mg2+ is inhibited. K(+)-activated p-nitrophenylphosphatase activity of the Cr(H2O)4AdoPP[CH2]P-inactivated enzyme decreases when an MgATP analogue, the tetraammine cobalt complex of ATP, Co(NH3)4ATP, is used additionally to inactivate the E2ATP site. The enzyme activity of K(+)-activated phosphatase is also lost if the beta,gamma-bidentate chromium(III) complex of ATP, Cr(H2O)4ATP, which may form a stable E1-chromo-phosphointermediate, is used for the inactivation of Na+/K(+)-ATPase. We conclude that the phenomenon of a blockade of the overall reaction of Na+/K(+)-ATPase by the formation of a stable E1.CrAdoPP[CH2]P complex, leading thereby to a loss of the partial activities of the E1 conformation, but not of the E2 conformation, is consistent with the postulate of an (alpha beta)2 diprotomeric nature of the sodium pump. The observation, moreover, that treatment of the sodium pump with Cr(H2O)4ATP but not with Cr(H2O)4AdoPP[CH2]P leads to an inactivation of K(+)-activated phosphatase seems to indicate that the formation of a E1-phosphointermediate affects the E2ATP site.

How do MgATP analogues differentially modify high‐affinity and low‐affinity ATP binding sites of Na+/K+ ‐ATPase?

The exchange-inert tetra-ammino-chromium complex of ATP [Cr(NH3)4ATP], unlike the analogous cobalt complex Co(NH3)4ATP, inactivated Na+/K(+)-ATPase slowly by interacting with the high-affinity ATP binding site. The inactivation proceeded at 37 degrees C with an inactivation rate constant of 1.34 x 10(-3) min-1 and with a dissociation constant of 0.62 microM. To assess the potential role of the water ligands of metal in binding and inactivation, a kinetic analysis of the inactivation of Na+/K(+)-ATPase by Cr(NH3)4ATP, and its H2O-substituted derivatives Cr(NH3)3(H2O)ATP, Cr(NH3)2(H2O)2ATP and Cr(H2O)4ATP was carried out. The substitution of the H2O ligands with NH3 ligands increased the apparent binding affinity and decreased the inactivation rate constants of the enzyme by these complexes. Inactivation by Cr(H2O)4ATP was 29-fold faster than the inactivation by Cr(NH3)4ATP. These results suggested that substitution to Cr(III) occurs during the inactivation of the enzyme. Additionally hydrogen bonding between water ligands of metal and the enzyme's active-site residues does not seem to play a significant role in the inactivation of Na+/K(+)-ATPase by Cr(III)-ATP complexes. Inactivation of the enzyme by Rh(H2O)nATP occurred by binding of this analogue to the high-affinity ATP site with an apparent dissociation constant of 1.8 microM. The observed inactivation rate constant of 2.11 x 10(-3) min-1 became higher when Na+ or Mg2+ or both were present. The presence of K+ however, increased the dissociation constant without altering the inactivation rate constant. High concentrations of Na+ reactivated the Rh(H2O)nATP-inactivated enzyme. Co(NH3)4ATP inactivates Na+/K(+)-ATPase by binding to the low-affinity ATP binding site only at high concentrations. However, inactivation of the enzyme by Cr(III)-ATP or Rh(III)-ATP complexes was prevented when low concentrations of Co(NH3)4ATP were present. This indicates that, although Co(NH3)4ATP interacts with both ATP sites, inactivation occurs only through the low-affinity ATP site. Inactivation of Na+/K(+)-ATPase was faster by the delta isomer of Co(NH3)4ATP than by the delta isomer. Co(NH3)4ATP, but not Cr(H2O)4ATP or adenosine 5'-[beta,gamma-methylene]triphosphate competitively inhibited K(+)-activated p-nitrophenylphosphatase activity of Na+/K(+)-ATPase, which is assumed to be a partial reaction of the enzyme catalyzed by the low-affinity ATP binding site.

A study of the vanadate-trapped state of the (Na,K)-ATPase. Evidence against interacting nucleotide site models.

The state of the Na+- and K+-stimulated adenosine triphosphatase ((Na,K)-ATPase) which binds vanadate was investigated by taking advantage of the slow rate of vanadate release (koff = 0.32/h at 25 degrees C and 0.045/h at 4 degrees C). Vanadate release from the enzyme could be accelerated approximately 2-fold by addition of K+ and 50-fold by addition of Na+. The K+ effect saturated hyperbolically with a K1/2 of 0.5 mM, while the Na+ effect had a sigmoidal activation curve and K1/2 of 250 mM. These results indicate that either Na+ or K+ can equilibrate with the vanadate-"trapped" enzyme prior to vanadate release. In the presence of vanadate, a saturable Mn2+ binding site could be detected with a dissociation constant of 120 nM. When 54Mn2+ was added during incubation of the enzyme with vanadate, 1 mol of 54Mn2+ could be trapped/mol of vanadate trapped and the two metals dissociated in parallel. This result indicates a single divalent cation site is involved in stabilizing vanadate (and probably phosphate) binding. Addition of 1 to 4 mM of ATP to the vanadate-trapped enzyme had no affect on the rate of vanadate release. Also, the high affinity ATP site could not be detected in equilibrium-binding studies with the vanadate-trapped enzyme. Since kinetic experiments indicate that vanadate binding is competitive with the low affinity ATP site (Cantley, L. C., Jr., Cantley, L. G., and Josephson, L. (1978) J. Biol. Chem. 253, 7361--7368) and the high affinity ATP site is absent on vanadate-trapped enzyme, it appears unlikely that ATP can occupy either a high or low affinity site on the vanadate-trapped enzyme. We show that nonlinear Lineweaver-Burk plots for ATP hydrolysis can be explained by a single hydrolysis site which exhibits a low affinity for ATP prior to the rate-limiting E2 to E1 conformational change, but a high affinity for ATP following the conformational change.

5'-p-fluorosulfonylbenzoyladenosine as an ATP site affinity probe for Na+, K+-ATPase.

We have investigated the suitability of 5'-p-fluorosulfonylbenzoyladenosine (FSBA) as an ATP site affinity probe for the canine kidney Na+, K+-ATPase. The purified enzyme is slowly inactivated by this compound in suitable buffers, losing about half of its activity over a two-hour period. The rate of inactivation is more rapid in 0.1 M KCl than in 0.1 M NaCl. Low concentrations of ATP protect the enzyme against inactivation, with half-maximal effects at 4 microM ATP in 0.1 M NaCl and 350 microM ATP in 0.1 M KCl. ADP also protects against FSBA inhibition, but AMP is ineffective when present at 100 microM levels. This pattern is consistent with the previously described nucleotide specificity of the Na+, K+-ATPase. Addition of protective amounts of ATP after inactivation has occurred does not restore enzyme activity, indicating that inhibition is irreversible. Measurement of the concentration-dependence of FSBA inactivation suggests an apparent Kd for binding of this compound well above 1 mM, the solubility limit of the analog. This finding is reinforced by the failure of 1 mM FSBA to compete effectively with ATP for the high-affinity ATP site of the enzyme. Nevertheless, attachment of the analog to this site is indicated by its ability to prevent [3H]-ADP binding in proportion to the number of sites it has inactivated. Studies with [3H]-FSBA show that about 1 mole of the analog attaches specifically to the alpha subunit per mole of enzyme inactivated. A similar amount of nonspecific labeling also occurs with negligible effect on enzyme activity. These findings suggest that FSBA may be useful in probing the topography of the high-affinity ATP binding site of the Na+, K+-ATPase and related enzymes.

Localization of the High‐Affinity ATP Site in Adenosine‐3′:5′‐monophosphate‐Dependent Protein Kinase Type I

8-Azido-adenosine 5'-triphosphate (n8(3)ATP) appeared to be a suitable photoaffinity label for the protein kinase dependent on adenosine 3':5'-monophosphate (cAMP). It competes with ATP for the high-affinity ATP site in the undissociated form of the kinase and in the phosphotransferase reaction catalyzed by the catalytic subunit. Furthermore, it is accepted as a substrate in the phosphotransfer reaction. n8(3)ATP incorporated into the holoenzyme is covalently bound irradiation. Protection experiments with ATP indicated that this covalent attachment occurs in the high-affinity ATP site of the enzyme. Polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate shows that n8(3)ATP is bound to the catalytic subunit. After irradiation the enzyme was dissociated by cAMP. Proportional to the incorporated [gamma-32P]n8(3)ATP, a loss in phosphotransferase activity was found. These results support our model that both ATP sites coincide with respect to their adenine binding part. Thus binding of the regulatory subunit to the catalytic subunit would then transform the low-affinity catalytically active ATP site into a high-affinity inactive site.

Mechanism of Activation of the Protein Kinase I from Rabbit Skeletal Muscle

The high-affinity binding site for ATP of the holoenzyme of cAMP-dependent protein kinase (type I) from rabbit skeletal muscle has been investigated. Binding affinity of a series of ATP derivatives substituted at different sites in the molecule was determined by competition with tritiated ATP. The results were compared with data available from cAMP derivatives with the same substituents, in order to analyse the electronic and steric features of these two sites on the protein kinase. The comparison revealed significant differences of the effect of substituents towards the two sites. In particular the N6-derivatives of ATP and substituents affecting the gamma-phosphate indicate that the high-affinity ATP site of the protein kinase has similar properties as those found for phosphotransferase sites. The present results are consistent with the supposition that the high-affinity site for ATP on the holoenzyme is congruent with the phosphotransferase site of the catalytic subunit. Upon combination of catalytic and regulatory subunits this site would be transformed into a high-affinity site for ATP with simultaneous blocking of the phosphotransferase activity.