Department of Chemistry, University of Toronto, 80 St George St., Toronto, Canada, M5S 3H6
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Abstract |
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Keywords: azobenzene/enzyme/photo-control/photoregulation/photoswitch
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Introduction |
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Perhaps the simplest reversibly photoisomerizable amino acid is phenylazophenylalanine (PAP) (Figure 1). It contains the photochromic unit azobenzene that can be switched between trans and cis conformations with appropriate wavelengths of light (Rau, 1990
; Nagele et al., 1997
; Wachtveitl et al., 1997
). In the dark, at equilibrium, azobenzene is predominantly trans (vide infra). Irradiation with ~340 nm light converts PAP from the trans to the cis isomer. The cis conformation slowly relaxes back to the trans conformation in the dark [
1/2
3 days (Zhang et al., 1999
)]. Alternatively, if the cis form is exposed to light of longer wavelength (>400 nm), photoisomerization to a predominantly trans conformation occurs rapidly. The reversibility of photoisomerization, the lack of significant side reactions (bleaching) and the compatibility of the wavelengths required with biological systems have made the azobenzene chromophore a popular photoswitch.
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We wished to perform a quantitative analysis of the effects of photoswitching in a site-specifically modified system to evaluate the strengths and limitations of this approach for controlling enzyme activity. We focus our analysis on photoregulation of the enzyme ribonuclease S (RNase S) since it has been the subject of several designed regulation attempts (Liu et al., 1997; Roy and Imperiali, 1997
; Hamachi et al., 1998
) and because the results can be assessed in the context of detailed high-resolution structural data (Richards et al., 1972
; Nogues et al., 1995
).
RNase S cleaves polymers of RNA on the 3' side of pyrimidine residues. It will also hydrolyse 2',3' ribonucleotide cyclic phosphates. RNase S is derived from ribonuclease A (RNase A) by the action of subtilisin (Richards and Wyckoff, 1971). Subtilisin cleaves RNase A between residues 20 and 21 to generate two components, the S-peptide (consisting of residues 120) and the S-protein (consisting of residues 21124) (Richards and Vithayathil, 1959
) (Figure 2
). There is no enzymatic activity observed in either the S-peptide or S-protein separately because each component contains only one of the two active site histidine residues (His 12 and His 119) that are required for enzymatic cleavage of the substrate (RNA) (Richards and Wyckoff, 1971
). When present together in solution however, the S-peptide and the S-protein form a non-covalent complex [Kd = 7.0x10-9 M (Richards and Wyckoff, 1971
)]. This complex is designated RNase S and displays essentially the same catalytic activity as RNase A (Richards and Vithayathil, 1959
). Chemical synthesis of analogues of the S-peptide containing PAP residues followed by recombination with S-protein provides a convenient means for generating RNase mutants containing PAP residues at specific sites. In general, however, PAP can be incorporated into proteins via tRNA-based suppression strategies (Hohsaka et al., 1994
; Kanda et al., 2000
); its incorporation does not rely on this fragment complementation approach.
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One would expect that substitutions of residues in groups (i) or (ii) with photoisomerizable amino acids would be most likely to lead to photomodulation of enzyme activity. We describe here the synthesis and kinetic characterization of RNase S' mutants carrying PAP residues in positions 4, 7, 8, 10, 11 and 13 of the S-peptide sequence. (The S' indicates that an analogue of the S-peptide has been incorporated into the active enzyme.) The locations of these residues in the three-dimensional structure of the enzyme are shown in Figure 2. The effects of isomerization on peptide binding to S-protein and on substrate binding and catalysis are quantitatively assessed.
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Materials and methods |
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Synthesis of Fmoc protected PAP was carried out according to Liu et al. (Liu et al., 1997) with modifications as described in Borisenko et al. (Borisenko et al., 2000
). Peptides were synthesized on a Pal-resin (capacity 0.55 mmol/g), 0.10 mmol scale (Advanced ChemTech, Louisville, KY). Coupling used 3 equivalents of O-(7-azobenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU); 6 equivalents of N,N-diisopropylethylamine (DIPEA); and 3 equivalents of the Fmoc protected amino acids (Ser(tBu), Asp(OtBu), His(Trt), Gln(Trt), Arg(Pbf), Glu(OtBu), Lys(Mtt), Thr(tBu)) (Advanced ChemTech). Peptides were cleaved from the Pal-resin and deprotected using a solution made up of 87% trifluoroacetic acid (TFA), 5% water, 5% thioanisole and 3% 1,2 ethanedithiol; total volume of the cleavage solution was 8 ml. The Pal-resin was removed by filtration and the peptides were precipitated in cold ether (35 ml). Peptides were dissolved in methanol and purified by preparative HPLC (22x250 mm, Apex ODS Prepsil 8U HPLC column, Jones Chromatography) using a Perkin-Elmer 250 LC binary pump HPLC system equipped with an UVVis detector. Mobile phases consisted of A: 0.1% TFA in acetonitrile and B: 0.1% TFA in water. A gradient program of 565% A over 30 min was used. The purity of all peptides was >95% (after HPLC) and all peptides had the expected mass by ESI-MS.
Photoisomerization of PAP
UVVis spectra of Fmoc-PAP and PAP peptide solutions were obtained using a Perkin-Elmer Lambda 2 spectrometer. The trans isomer was obtained by storing solutions [in 0.1 M 2-(N-morpholino) ethanesulphonic acid (MES) buffer, pH 6.0] in the dark at room temperature for >5 days. PAP samples were isomerized from trans to cis isomers using a Tri-Lite high intensity fibre optic light source (World Precision Instruments, FL) with a 340 ± 40 nm bandpass filter or a nitrogen laser ( = 337 nm, 150 µJ/pulse, 1.5 ns/pulse, 15 Hz) for ~20 min. Irradiation was continued until the UVVis absorbance spectra showed no further changes (530 min depending on solution concentration). Spectra of the isolated cis and trans isomers of Fmoc-PAP were obtained using an HPLC equipped with a photodiode array detector (Perkin-Elmer photodiode array detector 235C). A gradient of 60% A (40% B) 90% A (10% B), over 15 min (where A: 0.1% TFA in acetonitrile and B: 0.1% TFA in water) was used with a Zorbax SB C-18 analytical column (4.6x250 mm). Spectra of the isolated cis and trans forms of Fmoc-PAP were used to determine relative molar extinction coefficients for the two forms.
Enzyme kinetics measurements
The enzyme kinetic assay consisted of three components, the RNase S-protein (Sigma, type XII-PR), an S-peptide analogue (see Table I for peptide sequences) and a fluorescent substrate. These were combined in a buffer containing 0.10 M MES, 0.10 M sodium chloride, adjusted to pH 6.0 with a solution of 1 M sodium hydroxide (NaOH). For all experiments, the S-protein concentration was fixed at 9.125x10-11 M. This concentration was obtained by dilution of an S-protein solution whose concentration was determined spectrophotometrically using
280 = 8989 (Sherwood and Potts, 1965
). Stock S-protein solutions for kinetic runs also contained albumin (bovine serum albumin Type X, Sigma) in 100-fold excess and 0.01% Tween 20 detergent (by volume) (James and Woolley, 1998
). S-protein stock solutions were stored in a -20°C freezer in small aliquots. S-protein aliquots were removed from the freezer and allowed to warm to 25°C in a water bath prior to their use in a kinetics experiment.
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For each kinetic measurement, baseline fluorescence emission at 515 nm (excitation wavelength 475 nm) was recorded, then the kinetic run was initiated by adding a 50 µl aliquot of S-protein solution. After measuring the initial rate of change of fluorescence, a small volume (5 µl) of concentrated RNase A solution (0.1 M) was added to the sample and the maximum fluorescence recorded. Initial rates were converted to units of mol/l/s (M/s) by dividing the initial rate of change of fluorescence by the maximal fluorescence change for each sample and multiplying by the initial substrate concentration. The data was analysed using Igor Pro software (Wavemetrics, Lake Oswego, OR).
To ensure consistent activity of S-protein samples, each aliquot was tested in a standard assay. The standard was composed of 9.125x10-11 M S-protein, 8 µM substrate and 0.8 µM V-peptide. The initial rate of the standard had to be 45 ± 4x10-10 M/s otherwise the aliquot was discarded. Each S-protein aliquot provided one standard measurement and three kinetics measurements.
Scheme I shows the various interactions possible between chemical species in solution. where Pt and Pc refer to the trans and cis forms of the PAP peptide, respectively, S is the S-protein, R is substrate and X is product. (In general, it is not possible to obtain PAP peptide solutions consisting of only one photoisomer. Thus, both cis and trans isomers must be considered in the kinetic expression.) The concentrations of Pt and Pc (which can be controlled by light) do not change on the timescale of an enzymatic rate measurement since isomerization of PAP is slow in the dark [
1/2
3 days (Zhang et al., 1999
)]. We did not consider direct binding of substrate to S-protein in our analysis; this is expected to be a relatively weak interaction (Neumann and Hofsteenge, 1994
).
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![]() | (1) |
If one assumes that the equilibria between S-protein, S-peptide analogues and substrate are established rapidly compared to the rate of cleavage of bound substrate, and that product release is also fast compared to cleavage, then one can show that the initial rate can be expressed as:
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Enzyme kinetics: varying PAP peptide concentration
Initial rates (Vo) of substrate cleavage were measured for different concentrations of peptide keeping the substrate concentration (8.0x10-6 M) and the S-protein concentration (9.125x10-11 M) fixed. Trans-PAP peptide stock solution concentrations were determined spectrophotometrically using 330 = 24 000. Peptide concentrations (5120x10-7 M) were determined by dilution of peptide stock solutions. Trans-PAP peptide solutions were kept in amber vials to protect them from light.
To determine initial rates as a function of cis-PAP peptide concentration, a trans peptide solution was first irradiated for 30 min. A spectrum of the PAP peptide sample (200600 nm) was taken to confirm maximal trans to cis isomerization. The stock cis-PAP peptide solution was then kept under the light source for the duration of the experiment and aliquots of the solution were pipetted and mixed prior to the start of each run.
For each concentration of peptide, the initial rate was determined by fitting a straight line to the first 3045 s of the kinetic run (after addition of S-protein) using Igor Pro software. At least three independent measurements of initial rate were made for each concentration of peptide analogue.
If Scheme I describes the situation adequately, initial rate as a function of [P] (for [R] =Ro) is given by:
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Insufficient information is available from plots of Vo versus [P] at two different fc ratios to permit direct fitting of experimental data to Equation 3. Instead, we fitted the rate data to a single rectangular hyperbola (Equation 4
) to permit a qualitative initial screen for effects of photoisomerization (Table II
).
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![]() | (4) |
Enzyme kinetics: varying substrate concentration
Initial rate measurements were made for three non-natural RNase S' species (PAP 4, PAP 7, PAP 13 in both cis and trans conformations) as a function of substrate concentration. The concentration of S-protein (9.125x10-11 M) and the concentration of PAP peptide (PAP 4: 8.3 µM, PAP 7: 4.6 µM and PAP 13: 16.1 µM) were kept fixed. Solutions with different substrate concentrations (from 2 to 65 µM) were prepared in individual semi-micro cuvettes. The total volume in the cuvette, before addition of S-protein, was adjusted to 350 µl with MES buffer. Triplicate measurements of initial rate at each substrate concentration were performed. Initial rates were plotted against substrate concentration.
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Results and discussion |
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The cis and trans conformations of PAP differ in their molecular shape, solvent accessible surface area and dipole moment (Figure 1) (Robertson, 1939
; Brown, 1966
; Rau, 1990
; Borisenko et al., 2000
). Trans-PAP has a larger surface area, an elongated form and a dipole moment near zero. Cis-PAP has a smaller surface area, is highly twisted and has a dipole moment of ~3D. These differences were reflected in the relative retention times of the Fmoc-PAP photoisomers on a reverse-phase HPLC column (Figure 3A
). The trans isomer had a considerably longer retention time than the cis isomer. Spectra of pure trans and cis isomers were obtained by coupling a photo-diode array detector to the HPLC instrument. Relative molar extinction coefficients for the trans and cis isomers were calculated by normalizing the absorbance spectra of the individual isomers at an isosbestic point (285 nm) (Figure 3B
). UVVis absorbance spectra of the various PAP-containing S-peptide analogues and Fmoc-PAP were virtually superimposable for wavelengths >310 nm as expected. The largest difference in absorbance occurs near 350 nm where
trans/
cis = 9.65. This ratio provides an estimate of the maximum photo-conversion possible under steady-state illumination (if the quantum yields for cis
trans and trans
cis are similar) since it represents the largest difference in rates of excitation of trans and cis ground state isomers. As is evident from the HPLC traces in Figure 3A
, irradiation of PAP solutions carried out as described under Materials and methods results in conversion of a solution from 96% trans-PAP to 90% cis-PAP.
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The active site that is formed upon association of an S-peptide analogue and the S-protein is a broad cleft on the surface of the enzyme. Interaction with the substrate occurs over a significant area of the cleft. To characterize the photosensitive RNase S' mutants, we used a fluorescence resonance energy transfer (FRET) based assay of enzyme activity developed previously in our laboratory (James and Woolley, 1998). This assay employs a 9-mer single stranded RNADNA hybrid substrate for RNase (5'-d(AAAA)-rU-d(AAAA)-3') that is long enough to span the entire substrate-binding cleft. The substrate is combined in a 500:1 ratio with the fluorescent reporter 5'(fluorescein)-d(AAAA)-rU-d(AAAA)-3'(rhodamine) (James and Woolley, 1998
). FRET occurring between the fluorescein and the rhodamine dyes ceases (and fluorescein emission is thereby enhanced) upon substrate cleavage. The fluorescent and non-fluorescent substrates were shown to have identical kinetic constants with RNase A in previous work (James and Woolley, 1998
).
This assay provides a sensitive measure of the ribonuclease activity that does not require large amounts of substrate or protein. By systematically varying the peptide component and the substrate component of the assay mixture, initial rate measurements can lead to a determination of the kinetic parameters of the reconstituted RNase S' (vide infra). By performing these measurements with PAP peptides in cis or trans conformations, the effects of photoisomerization on enzyme activity can be quantitatively assessed.
We synthesized a non-photosensitive S-peptide analogue (V-peptide, residues 415, M13V) to be used as an internal standard to ensure consistent activity of the RNase S' system during characterization of all of the PAP peptide analogues. The methionine in position 13 was mutated to a valine (hence V-peptide) to avoid potential problems with methionine oxidation (Goldberg and Baldwin, 1998). This mutation does not substantially affect the binding or activity of the peptide (Varadarajan et al., 1992
). Initial rates measured with different S-protein preparations were checked for consistency using standard solutions of the V-peptide.
Kinetic characterization of the effects of PAP mutations
The PAP peptides discussed here are numbered to correspond to native S-peptide nomenclature (e.g. PAP 7 is a truncated S-peptide analogue with PAP at position 7 of the native S-peptide sequence). Peptides were made with PAP in positions 4, 7, 8, 10, 11 and 13 (Table I). Amino acids in positions 4, 7, 10 and 11 were selected because mutation at these positions would place PAP on the surface of the substrate-binding cleft of RNase S' (Richards and Wyckoff, 1971
; Blackburn and Moore, 1982
; Raines, 1998
) (Figure 2
). The residues in positions 8 and 13 (Phe and Met, respectively) are important in S-protein and S-peptide interactions (Richards and Wyckoff, 1971
; Blackburn and Moore, 1982
) (Figure 2
). It should be noted that the PAP 13 peptide consisted of residues 115 of the native sequence, rather than residues 415 as with the other PAP peptides. We used the longer peptide sequence for PAP 13 in order to compare our results with those reported by Hamachi et al., who also investigated photoregulation of RNase S' with this peptide (Hamachi et al., 1998
) (vide infra).
Under the conditions of these experiments, cis PAP peptide solutions were 90% cis/10% trans and trans PAP peptide solutions were 96% trans/4% cis (Figure 3). Azobenzene can be converted to >99% trans by prolonged storage in the dark at elevated temperatures (Renner et al., 2000
); however, this is rather impractical as a general strategy since enzyme denaturation might be expected. Conversion to >90% cis is not possible because there is no wavelength where the difference in UV absorbance between the cis and trans isomers [Figure 3B
(inset)] is larger than ~10-fold.
Given the ratios of cis and trans species observed, one can use Equation 2 with arbitrary choices for the kinetic constants to calculate rates for cis and trans samples for a matrix of peptide and substrate concentrations. If the cis form is more active than the trans (e.g. if kct = 0 or Kst is very large), a maximum difference in initial rate of 22.5-fold (Vo(cis)/Vo(trans)) is possible. If the trans form is more active, a 9.6-fold difference (Vo(trans)/Vo(cis)) is possible. These values are simply the relative change in concentration of the active isomer. Of course, much smaller rate differences are possible even with large changes in individual constants upon photoisomerization. For instance, a decrease in peptide affinity for the S-protein may be offset by an increase in the affinity of substrate for the active site.
Effects of individual mutations
When the PAP 8 peptide was studied, no activity was observed in either the cis or trans conformation. This corroborated observations originally reported by Liu et al. and later by Hamachi et al. (Liu et al., 1997; Hamachi et al., 1998
) and can be understood in terms of the three-dimensional structure of the protein which provides a tight binding pocket that cannot accommodate extensions to the Phe 8 side-chain (Richards and Wyckoff, 1971
; Blackburn and Moore, 1982
) (Figure 2
).
For PAP peptides 10 and 11 the apparent maximum rate (Va) for the cis and trans forms were the same within experimental error. The apparent Ka values differed measurably but the differences were small (<2-fold in each case).
While no effect on the parameters Va and Ka does not rule out an effect of photoisomerization on one or more of the constants in Equation 2, we elected to focus on peptides that showed the largest effects on Va and Ka, i.e. PAP 4, PAP 7 and PAP 13 (Table II
). [While photoisomerization could have an effect on kc (for example) that compensated for its effect on Ks, a simpler explanation would be that photoisomerization affects neither kc nor Ks significantly.] These RNase S' species were selected for further characterization by measuring initial rates as a function of substrate concentration and carrying out a global analysis to obtain all the constants defined in Scheme I
for each isomer. The fitted curves are shown for PAP 4 (Figure 4
), PAP 7 (Figure 5
) and PAP 13 (Figure 6
) and the calculated constants are collected in Table III
.
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When compared to native RNase A, both PAP 4 peptides (cis/trans) show about a 2-fold decrease in kc and a 4-fold weakening of substrate binding (Ks). This observation is consistent with previous studies on the PAP 4 enzyme using a cyclic nucleotide substrate that indicated near wild-type activity (Liu et al., 1997). The absence of a large effect on kc is consistent with the location of the PAP residue a considerable distance from the site of bond cleavage and with relatively minor effects on the conformation of the bound PAP peptide and substrate. Likewise, photoisomerization of PAP 4 does not appear to affect the conformation of the bound PAP peptide and substrate substantially since the kc and Ks parameters are hardly affected. The largest effect of photoisomerization for the PAP 4 system appears to be on the dissociation constant of the peptide from S-protein (Kd), which changes ~3-fold. PAP 4 is located at the N-terminal end of the reconstituted RNase S [it replaces Ala 4 (Figure 2
)], and does not appear to make strong interactions with other parts of the S-protein (Figure 2
). The mechanism whereby isomerization of the PAP residue affects binding affinity may be subtle, for instance it may affect the helical propensity of the S-peptide (Goldberg and Baldwin, 1999
).
PAP 7 RNase S'
The PAP 7 substitution replaces a Lys normally at that position. Lys 7 forms part of the P2 subsite that interacts with the phosphate backbone of the RNA substrate (Boix et al., 1994; Fisher et al., 1998
; Raines, 1998
). When Lys 7 was replaced by an uncharged or a negatively charged amino acid (e.g. Ala, Glu) decreases in the rate of hydrolysis of dinucleotide and polymer substrates were observed (CpA and poly C) (Boix et al., 1994
; Fisher et al., 1998
). In the present case, kc for cis or trans PAP 7 RNase S' is decreased ~5-fold compared to RNase A whereas Ks is only marginally affected. Isomerization of PAP 7 between cis and trans forms causes small changes in each of kc, Ks and Kd (Table III
). The largest of these is a 3-fold change in Kd, however the effects all operate in the same direction. That is, for a given PAP 7 peptide and substrate concentration the cis isomer will generally be less active than the trans isomer because the cis-peptide binds more weakly to the S-protein, the substrate binds more weakly to cis-PAP 7 RNase S' and the chemical step is less efficient for the cis-PAP 7 RNase S' enzyme. This is in contrast to the PAP 4 case where, for instance, an increased kc for the cis case tends to counteract a decreased affinity of the cis PAP 4 peptide (Table III
).
PAP 13 RNase S'
The PAP 13 substitution, which replaces Met 13, had substantially reduced peptide-binding affinity compared to PAP 4, 7, 10 and 11 but did remain active (Table III). The data do not permit separate determination of Kdc and Ksc (the peptide and substrate dissociation constants for this cis form) with statistical significance, but only their product (KdcKsc), which is similar to the trans form. With trans PAP 13, the dissociation constant was significantly larger than for PAP 4 and 7. This occurred despite the presence of residues 13 in the PAP 13 peptide, which are expected to contribute substantially to the binding affinity. The apparent affinity of S-peptide 120 is ~1 nM versus 3 µM for S-peptide 420 (Richards and Wyckoff, 1971
; Blackburn and Moore, 1982
; Komoriya and Chaiken, 1982
). The reduced binding ability of PAP 13 is also consistent with previous work on (non-photoisomerizable) mutations at position 13 of RNase S (Blackburn and Moore, 1982
; Varadarajan et al., 1992
; Thomson et al., 1994
) and with the location of this residue in the RNase S' structure (Figure 2
). The Met residue normally in this position makes contacts with the S-protein that contribute substantially to the binding energy of the S-peptideS-protein interaction (Varadarajan et al., 1992
; Thomson et al., 1994
). The rate constant of the chemical step (kc) is 10-fold less than wild-type for trans-PAP 13 RNase S' and 20-fold less for cis-PAP 13 RNase S'. PAP 13 RNase S' thus exhibits the most perturbed kinetic behaviour of RNase S' species studied here.
Hamachi et al. (Hamachi et al., 1998) reported `almost all-or-none type' photoregulation of RNase S' using the same PAP 13 peptide and a UV absorbance assay (delCardayre and Raines, 1994
). Under the conditions of their experiment, trans-PAP 13 RNase S' was active whereas the rate of cis-PAP 13 RNase S' was too low to measure. Details of the assay (i.e. peptide, protein or substrate concentrations) were not provided; however, the concentrations of all components were presumably considerably higher than in the present case since UV absorbance measurements provide a relatively insensitive measure of RNase activity. Using the kinetic constants determined for PAP 13 RNase S' (Table III
) and Equation 2
, we calculated the ratio of initial rates (Vo(trans)/Vo(cis)) to be expected for a matrix of [P] and [R] concentrations. Consistent with the observations reported by Hamachi, we find that the trans form of PAP 13 RNase S' is more active than the cis form; however, the ratio Vo(trans)/Vo(cis) never exceeds ~2.3. Thus, the cis form is expected to be, at most, ~2-fold less active than the trans forma result that appears inconsistent with the data reported by Hamachi et al. unless the rate of the cis form is simply slightly below their detection limit. Indeed, as pointed out above, because of incomplete photo-conversion, a difference in rate of ~10 fold (Vo(trans)/Vo(cis)) is the maximum possible. Perhaps the inactivity of the cis isomer reported by Hamachi et al. is the result of another effect, for instance cis-PAP 13 RNase S' aggregation at higher concentrations (Gotte et al., 1999
; Park and Raines, 2000
), that somehow amplifies the effects of photoisomerization.
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Conclusions |
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While the results confirm that the site of placement of the photoisomerizable group is important (e.g. the MetPAP 13 mutation has a marked effect on peptide binding), differences between cis and trans isomers tend to be small and distributed between effects on peptide binding, substrate binding and the rate constant for the chemical step. Simultaneous optimization of effects of photoisomerization on these steps might best be accomplished via a combinatorial approach.
The fact that most PAP mutations examined led only to minor effects on kinetic constants implies that isomerization of a PAP residue, even one placed in close proximity to an enzyme active site will not, in general, cause large changes in enzyme behaviour. Perhaps the flexibility of the PAP residue is sufficient to mask the conformational differences that result from isomerization of the azo bond (Zhang et al., 1999). Alternatively, other side chain movements in the enzyme may accommodate the conformational changes of the PAP side chain. This effect might be particularly true for RNase S, which acts on a flexible polymeric substrate. Larger effects might be observed with more rigid active sites or if the isomerization caused a more substantial structural change. Larger effects might also be seen if the conformational change upon photoisomerization could be coupled to a larger conformational change within the protein. Azobenzene-based crosslinkers have recently been shown to affect the degree of helicity of short water-soluble peptides (Kumita et al., 2000
). A similar strategy might be used to photoregulate the conformation of an S-peptide analogue and thereby regulate RNase S' activity.
Finally, the incomplete photo-conversion of PAP between cis and trans isomers sets intrinsic limits on the degree of photomodulation of enzyme activity that might be possible using this photochromic amino acid. While changes in activity of 1020-fold might be sufficient for certain applications, larger changes would make photoswitchable proteins more desirable tools. Azobenzene derivatives that undergo thermal relaxation more quickly (Kumita et al., 2000) would make production of >99% of the trans form practical. Larger effective changes in the concentration of the cis form upon irradiation could then be achieved.
Another possible strategy for achieving larger changes in the concentration of the active isomer may be to employ multiple PAP residues for photoregulation (Lien et al., 1996). If only the cis/cis isomer were active in an enzyme system containing two PAP residues and the cis/trans, trans/cis and trans/trans isomers were all inactive then a switch from 4% cis (the trans sample) to 90% cis (the cis sample) would result in a 500-fold change in the concentration of the active isomer. An analogous strategy would be to attempt photoregulation of an enzyme system that required dimerization or oligomerization for activity. If photoisomerization caused a change in the association constant between domains in an oligomeric complex, a limited degree of isomerization might be substantially amplified.
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Notes |
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Acknowledgments |
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References |
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Received May 20, 2001; revised August 13, 2001; accepted September 10, 2001.