(Received for publication, May 21, 1996, and in revised form, September 23, 1996)
From the Department of Chemistry, Columbia University, New York, New York 10027
Escherichia coli tRNA pseudouridine synthase I (PSUI) catalyzes the conversion of uridine residues to pseudouridine in positions 38, 39, and 40 of various tRNA molecules. In previous biochemical studies with this enzyme (Kammen, H. O., Marvel, C. C., Hardy, L., and Penhoet, E. E. (1988) J. Biol. Chem. 263, 2255-2263) it was reported that cysteine residues are important in maintaining the active structure of the enzyme and are possibly involved in the catalytic reaction mechanism via a covalent cysteine intermediate. In order to further investigate the biochemical properties of PSUI, a high level expression and purification system for the enzyme and its corresponding mutants was developed. PSUI has three cysteine residues among 270 amino acids. In the present investigation, each cysteine residue was individually changed to serine and alanine. In addition, a triple mutant was prepared wherein all three cysteine residues were replaced by alanine. Surprisingly, while two of the three cysteine to serine mutants were inactive, all alanine mutants exhibited near wild-type levels of activity, including the triple mutant. These results provide the first direct and unambiguous chemical evidence against a covalent cysteine intermediate in the rearrangement mechanism of uridine to pseudouridine.
RNA is unique among nucleic acids for its numerous modified
nucleotide bases. To date, there have been approximately 93 chemically distinct modified bases identified in various RNAs (1). Among them,
pseudouridine () is the most common modified nucleotide present in
terms of distribution and frequency of occurrence (Fig. 1). It is present in all organisms ranging from
prokaryotes to mammals and is found in transfer RNA (tRNA), ribosomal
RNA (rRNA), and small nuclear RNA (snRNA) (2-4). Pseudouridine and its
N1- and/or N3-derivatized analogs
comprise the only carbon nucleotides (carbon-carbon glycosyl bond) so
far identified at the polynucleotide level.
The formation of the widely distributed group of pseudouridines is due
to a collection of RNA pseudouridine synthases that have a high degree
of site specificity. Escherichia coli tRNA pseudouridine
synthase I (PSUI)1 is one of the few
synthases whose gene (truA) has been
identified (5).2 It catalyzes the formation of in
positions 38, 39, and 40 in tRNA molecules (6). The biosynthesis of
takes place at the polynucleotide level and involves an intramolecular
rearrangement of U (7-10). Mechanistically, not much is known about
the rearrangement, but neither ATP nor cofactors are required (8).
Examination of the structure of
suggests that it is chemically
formed from U by first cleavage of the carbon-nitrogen glycosyl bond,
followed by a 180° flip (or 120° rotation) of the uracil base and
then reattachment at C5 to yield
. The overall process
can be viewed as a combination of enzymatic events having mechanistic
similarities to uracil glycosylases and DNA/RNA methyltransferases. In
formation, the cleavage of uracil from ribose bears analogy to the
action of uracil glycosylases, while the reattachment step represents
an alkylation event similar to that seen for methyltransferases. A well
known mechanistic step in certain enzymatic processing of nucleic acids
involves a general Michael addition type mechanism to either uracil or
cytosine (11). This is observed for thymidylate synthase (12), dUMP,
and dCMP hydroxymethylases (13), DNA (cytosine-5)-methyltransferases
(14, 15), and tRNA (m5U54) methyltransferase (16, 17). In
addition, reversible Michael adducts have been implicated in the
interaction between aminoacyl tRNA synthetases and cognate tRNAs (18,
19). In all cases, the nucleophile in the Michael addition step is the
thiol from a cysteine residue of the enzyme. Attack at C6
of the pyrimidine ring forms the covalent cysteine intermediate, which,
in the case of methyltransferases, results in activation at
C5 for electrophilic attack. Since the late 1970s, an
analogous Michael addition type mechanism has been postulated to be
also involved in
formation (10, 11, 17-20). The reaction is
similar to that of methyltransferases, since both involve an alkylation reaction at C5 of U. In one case the alkylation is
intermolecular, while in the other, it is intramolecular. In addition
to these reaction similarities, further indication for the possible
involvement of a Michael type process was provided by the result that
the acid-catalyzed hydrolysis of U proceeds through the addition of water across the 5,6-double bond to form a 5,6-dihydrouridine intermediate (21).
The first biochemical evidence implicating the importance of cysteine
residues in the catalysis of formation was reported by Kammen
et al. (10) using recombinant PSUI. In that study, enzymatic
activity was dramatically impaired in the presence of thiol-specific
modification reagents such as p-chloromercuribenzoic acid
(pCMB), iodoacetate, or 5,5
-dithiobis-(2-nitrobenzoic acid). A similar
reduction in activity was observed when reducing thiols were omitted
from the reaction buffer. Furthermore, PSUI activity was inhibited by
tRNAs containing 5-fluorouridine (5-FU). A mechanism consistent with
the above observations was advanced that invoked the use of a
nucleophilic cysteine residue (10). Attack of the uridine
C5-C6 double bond by SH of cysteine results in
a covalent intermediate at C6 (Fig. 2). This
would be followed by carbon-nitrogen glycosyl bond cleavage, base
rotation, and formation of the carbon-carbon bond between
C5 of uracil and C1
of ribose. Elimination of
the covalent enzyme-substrate intermediate affords
and regenerates
the enzyme. An attractive feature of this mechanism is the utilization
of cysteine, which not only facilitates activation at C5
toward alkylation but also serves to "hold on" to the uracil base
for reattachment after its initial cleavage.
E. coli pseudouridine synthase I is a 31-kDa protein that
contains three cysteine residues among 270 amino acids. The U to rearrangement catalyzed by this enzyme is chemically intriguing, since
very little is known about the mechanistic steps involved in the
transformation. In order to further investigate the biochemical properties of PSUI, a high level expression and purification system for
the enzyme and its corresponding mutants was developed. In particular,
the role of each cysteine residue in maintaining catalytic activity was
specifically probed by site-directed mutagenesis. Much to our surprise,
and contrary to the long held notion, the results indicate that the
rearrangement not only proceeds without a covalent cysteine
intermediate but that it also does not require cysteine residues for
activity.
General biochemical reagents and buffers were
purchased from common commercial vendors. Radioactive triphosphates
were obtained from Amersham. [5-3H]UTP was purchased from
Andotek (Irvine, CA). Mature yeast tRNAPhe was purchased
from Sigma. Restriction endonucleases and T4 DNA ligase were purchased from New England Biolabs. Taq DNA
polymerase was purchased from Perkin-Elmer. Ribonucleotide
5-triphosphates were purchased from Pharmacia Biotech. Aniline
(99.5%) and pCMB were purchased from Aldrich and used without further
purification. T7 RNA polymerase was isolated from the E. coli strain BL21/pAR1219 and purified by the method of Wyatt
et al. (22). Plasmid pNU61, which carries the coding
sequence of the PSUI gene, was a kind gift from Professor Malcolm
Winkler. Expression vector pET3d and BL21(DE3) pLysS strain were
obtained from Novagen. DNA manipulation and transformation were
performed using methods described by Maniatis et al. (23).
Restriction digests and ligations were performed using conditions
recommended by the enzyme suppliers. Oligonucleotides were made on an
Applied Biosystems 391 DNA synthesizer and purified by 15% denaturing
polyacrylamide gel electrophoresis. DNA sequencing was performed using
the Sequenase kit from U.S. Biochemical Corp.
Primers
containing the partial coding sequence of PSUI with flanking
NcoI and BamHI restriction sites were used in the
polymerase chain reaction (PCR) to replicate the coding sequence of
PSUI from pNU61. A 100-µl reaction mixture contained 50 mM KCl, 10 mM Tris-HCl, pH 8.3, 3.75 mM MgCl2, 0.2 mM each of the
deoxyribonucleotide triphosphates (dATP, dCTP, dGTP, and TTP), 25 pmol
each of the primers P1 and P2, 3 ng/ml pNU61, and 2.5 units of
Taq DNA polymerase. P1 was
5-CTAGCCATGGTA
-3
and P2
was 5
-ATCTGGATCC
-3
. The
underscored sequences are PSUI sequences, and restriction sites are
italicized. The annealing temperature used was 55 °C. After
amplification, the resulting DNA was digested with NcoI and
BamHI, and the fragment was subcloned into the
NcoI and BamHI sites of pET3d vector. Both
strands of the resulting clone were sequenced. The construct, plasmid
pDH101, was transformed into E. coli BL21(DE3) pLysS strain
and grown in NZYM medium containing ampicillin and chloramphenicol. At
midlog phase, the cells were induced with 0.2 mM isopropyl
-D-thiogalactoside for 3.5-4 h and were collected by
centrifugation.
The induced cell pellet from a 1-liter culture was resuspended in 10 ml of TEM buffer (10 mM Tris-HCl, pH 8.0, 10 mM MgCl2, 0.1 mM EDTA), and lysed in a French pressure cell. The lysate was centrifuged at 65,000 × g for 1 h at 4 °C. The supernatant was loaded onto a DEAE-cellulose column (28 × 200 mm) preequilibrated with TESG buffer (20 mM Tris-HCl, pH 7.5, 0.1 mM EDTA, 5 mM 2-mercaptoethanol, 10% glycerol). The column was eluted with 200 ml of a 0-0.8 M NaCl gradient in TESG buffer. Peak fractions (30 ml) were collected between 0.2 and 0.4 M NaCl and dialyzed against MESG buffer (20 mM MES, pH 6.5, 0.1 mM EDTA, 5 mM 2-mercaptoethanol, 10% glycerol) and then loaded onto a phosphocellulose column (25 × 100 mm) preequilibrated with MESG buffer. The column was eluted with 200 ml of a 0-0.9 M NaCl gradient in MESG buffer. Peak fractions (30 ml) were collected between 0.6-0.7 M NaCl. Solid ammonium sulfate was added to give a final concentration of 60% (w/v). The mixture was centrifuged at 40,000 × g for 30 min at 4 °C. The pellet was dissolved in TESG buffer and dialyzed against TESG buffer followed by 1:1 TESG buffer:glycerol. The final yield of purified PSUI was approximately 20 mg.
MutagenesisIn vitro mutagenesis was performed
by overlap extension site-directed mutagenesis using the polymerase
chain reaction as described previously (24). PCR primers used for the
generation of mutants were as follows: M(C55S),
5-CCGTCTTCTCCGCCGGGCGTA-3
and 5
-TACGCCCGGCGGAGAAGACGG-3
; M(C154S),
5
-GCTGCGCAATCCTTGCTGGGCGA-3
and 5
-TCGCCCAGCAAGGATTGCGCAGC-3
; M(C169S), 5
-GCGGTGCAGTCCCAGTCCCGAA-3
and
5
-TTCGGGACTGGGACTGCACCGC-3
; M(C154A), 5
-GCTGCGCAAGCCTTGCTGGGCGA-3
and 5
-TCGCCCAGCAAGGCTTGCGCAGC-3
; M(C169A),
5
-GCGGTGCAGGCCCAGTCCCGAA-3
and 5
-TTCGGGACTGGGCCTGCACCGC-3
; M(C55A),
5
-CCGTCTTCGCCGCCGGGCGTA-3
and 5
-TACGGGGGGCGGCGAAGACGG-3
. The
two flanking PCR primers were the same as those used for subcloning. The conditions for PCR amplification and the subcloning into the pET3d
vector were the same as described above for construction of the
wild-type expression vector. Overexpression and purification of mutant
proteins were analogous to wild-type enzyme. Yields ranged from 2 to 20 mg of purified protein/liter of induced cells.
Plasmid pETRP13 containing the E. coli tRNAPhe gene was digested with BstNI to produce linear templates for in vitro transcription. Transcription reactions of [5-3H]UTP-labeled tRNAPhe were carried out at 37 °C for 90 min in a 100-µl reaction volume containing 0.1 mg/ml DNA template, 0.5 mM each of ATP, CTP, and GTP, 0.1 mCi/ml [5-3H]UTP, 6 mM MgCl2, 40 mM Tris, pH 7.9, 10 mM DTT, 2 mM spermidine, 0.01 units/ml RNasin, 3 units/ml T7 RNA polymerase. Reactions were stopped by the addition of EDTA to 50 mM. The mixture was then passed through a Sephadex G50 spun column to remove unincorporated triphosphates. Transcripts were incubated in a buffer containing 50 mM Tris, pH 8.0, and 10 mM MgCl2 for 60 min at 37 °C to renature tRNAPhe in its native form. Tritium-labeled transcripts were quantified by liquid scintillation counting.
Activity AssayThe tritium release assay used in this work was done as described previously with some modifications (8). In general, reactions were done in a 200-µl volume containing 50 mM Tris, pH 8.0, 10 mM MgCl2, 10 mM DTT, 0.1 mg/ml bovine serum albumin, 5 nM [3H]tRNA, and 5 nM enzyme. After incubating at 30 °C for 20 min, reactions were stopped by the addition of 300 µl of 15% Norit A in 0.1 N HCl. After standing at room temperature for 5 min, the activated charcoal was removed by centrifugation. The mixture was filtered through a 0.45-µm filter and washed twice with buffer, and the resulting filtrate was counted using a liquid scintillation counter.
Examination of Sulfhydryl RequirementThe thiol-specific reagent, pCMB, was used to check the thiol dependence of PSUI. The enzyme (5 nM) was preincubated with pCMB (0.25 mM) at 30 °C for 15 min followed by addition to the reaction mixture (minus DTT). The extent of the reaction was monitored after 20 min. To examine the reversibility of pCMB inactivation, DTT (10 mM) was added to the reaction after 20 min, and the reaction was then allowed to proceed for an additional 20 min.
Kinetic StudiesKinetic studies were performed using the tritium release assay with wild-type PSUI and triple mutant M(AAA) enzyme. Kinetics were carried out in 200-µl reactions. PSUI concentration was 1 nM and tRNAPhe concentrations ranged from 20-200 nM. The ratio of unlabeled and tritium-labeled tRNAPhe transcripts was 99:1. Initial velocities were determined by linear regression of DPM versus time plots. Data were fit to the Michealis-Menten equation, and the kinetic parameters were determined. The reported kinetic parameters represent the average of three separate determinations with correlation coefficients of 0.98.
The previously known
plasmids, pNU61 and 300 containing the truA
(hisT) gene sequence of PSUI were formerly the best
available source for recombinant PSUI (6, 10). In this system, PSUI is
under the control of its natural promoter and is produced in less than
0.2% of total soluble protein. In order to further study the
biochemical properties of this enzyme, initial efforts were directed at
creating a high level PSUI expression system. The PSUI coding sequence
was amplified by PCR using pNU61 as template. Two extra amino acids
(methionine and valine) were added in front of PSUI in order to create
the correct reading frame. The PCR product was digested with
NcoI and BamHI and subcloned into the corresponding sites of pET3d to give pDH101. Here, PSUI is under control of the T7 promoter. Upon induction with 0.2 mM
isopropyl
-D-thiogalactoside, the major component of the
soluble cell extracts was overexpressed PSUI. The expression levels and
purification results are shown in Fig. 3. Approximately
20 mg of pure PSUI can be obtained from 1 liter of induced cells. All
mutants were expressed and purified analogously to the wild-type
protein, affording 2-20 mg/liter.
Activities of Mutant Enzymes
Based on prior biochemical
evidence implicating the participation of cysteine residues in the
catalysis of U to (10), the role of these cysteines was
specifically examined by site-directed mutagenesis. E. coli
PSUI has three cysteine residues located at positions 55, 154, and 169. Each cysteine was individually changed to a serine to generate mutant
proteins, M(C55S), M(C154S), and M(C169S). The activities of these
mutants were tested, and the results are presented in Table
I. While M(C55S) maintained activity levels similar to
those of wild-type enzyme, M(C154S) and M(C169S) possessed little to no
enzymatic activity. To further examine the function of these residues,
a similar series of mutants were prepared in which cysteine was changed
to the nonpolar residue alanine. Three mutants were made wherein Ala
replaced Cys in positions 154 (M(C154A)), 169 (M(C169A)), and in all
three locations 55, 154, and 169, which afforded the triple mutant,
M(AAA). The activities of these mutants were tested, and the results
are presented in Table I. In contrast to the Ser mutants, all Ala
mutants showed high levels of activity, even the triple mutant, which
has all three Cys replaced by Ala.
|
Kammen and co-workers (10) previously investigated the thiol dependence of PSUI on catalysis. It was reported that the thiol-specific reagent, pCMB, irreversibly inactivated the enzyme. Table II shows the inactivation of PSUI in the presence of 0.25 mM pCMB, but contrary to the previous report, the inhibition was reversible upon the addition of 10 mM DTT. Further, the activities of Ala mutants, M(C154A), M(C169A), and M(AAA) were also examined in the presence of 0.25 mM pCMB (Table II). All proteins showed a dramatic impairment of activity in the presence of pCMB (minus DTT) except the triple mutant, M(AAA), which lacks Cys residues.
|
activity of wild-type and triple mutant M(AAA) enzymes were measured as
a function of increasing substrate concentration (Fig.
4). Initial velocities were plotted against substrate
concentration, from which Vmax and
Km were determined. Vmax
values for wild-type and M(AAA) enzymes are 380 ± 24 and 240 ± 28 pM s
1, respectively.
Km values for wild-type and M(AAA) enzymes are
86 ± 14 and 110 ± 27, respectively. Replacement of all
three Cys residues with Ala in M(AAA) resulted in only a small change in both Km and Vmax
parameters as compared with wild-type enzyme. These mutations caused a
1.2-fold increase in Km and a 1.6-fold decrease in
Vmax. The overall
Vmax/Km ratio was lowered by
a factor of 2 for the triple mutant, M(AAA).
A compilation of E. coli tRNA sequences reveals residues at seven different positions, namely 13, 32, 38, 39, 40, 55, and 65 (1-4). There are nine identifiable positions in which
is found in E. coli rRNA (4). E. coli PSUI
specifically catalyzes the conversion of U to
in positions 38, 39, and 40 in tRNA (6). This particular modification is highly dependent on
the tertiary structure of the tRNA.3 During
the course of the present study, Ofengand and co-workers cloned the
genes for several
synthases from E. coli.
55 synthase (truB) specifically modifies U to
at position 55 in tRNA
(26).
746 synthase (rluA) modifies both positions 32 in
tRNA and 746 in 23 S rRNA (27). And finally,
516 synthase
(rsuA) modifies position 516 in 16 S rRNA (28).
Surprisingly, although all
synthases catalyze the same type of
reaction in RNA, wherein U is rearranged to
, there is no obvious
sequence homology between these enzymes (27). All of these synthases
contain two or three cysteine residues, but there is no conserved
alignment of these residues in their primary sequence.
Previous studies on formation suggested that the mechanism of this
reaction involves cleavage of the uracil base followed by reattachment
of the base to the ribose at C5 of uracil (7-10). A well
established mechanistic pathway of enzymes that effect reaction at
C5 of pyrimidines (i.e. methyltransferases)
utilizes covalent cysteine intermediates via a Michael type addition
(11-17, 20). Similarly, cysteine residues have been implicated to play
an important role in the catalyzed rearrangement of U to
by PSUI
(10). Synthase activity was shown to be irreversibly inactivated by the
addition of pCMB (10). This inactivation has been verified in the
present study, but in contrast to the previous report, the inactivation was found to be reversible. A similar reversibility has been reported for tRNA (m5U54) methyltransferase (16). On the other hand,
a previous study showed that while various
synthase activities were
inhibited in the presence of 5-FU tRNAs, no covalent intermediates
could be detected (29). This result, however, may very well be a
consequence of the underlying mechanism of the transformation and not
the absence of a covalent intermediate. Fig. 5
illustrates this point. It is conceivable that while 5-FU residues have
been used with great success in trapping covalent cysteine
intermediates in methyltransferases (12, 16), they may not be
applicable for studying
formation in the same manner, due to the
difference in their reaction pathways. Since the rearrangement of U to
results in a free N1-H imino proton,
-elimination of
a covalent intermediate at C6 could conceivably take place.
This would release the enzyme from its substrate, and thus prevent
trapping of a covalent adduct. Such an elimination pathway is not
available in the mechanism of U or C methylation by
methyltransferases.
In order to clarify these mechanistic ambiguities, the participation of
cysteine residues in maintaining synthase activity was directly
investigated by site-directed mutagenesis. Initially, serine was chosen
to replace cysteine, since structurally and functionally serine is
comparable with cysteine. The three cysteine residues of PSUI located
in positions 55, 154, and 169 were each individually mutated to serine.
An E. coli tRNAPhe transcript containing uridine
residues labeled with 3H at C5 was used as the
substrate. This tRNA contains a modifiable U at position 39, which is
transformed to
when exposed to PSUI.3 The extent of the
U to
rearrangement was monitored by the tritium release assay (8).
This assay takes advantage of proton release from C5 during
the course of the U to
transformation. Monitoring
3H+ release from a tritium-labeled substrate
provides a relatively easy method for measuring enzyme activity. While
mutant M(C55S) had little effect on activity in comparison with
wild-type PSUI, mutants M(C154S) and M(C169S) showed virtually no
activity, thus indicating that these mutations severely impaired
catalytic function (Table I). Both mutants M(C55S) and M(C154S) were
capable of binding tRNAPhe as assayed by electrophoretic
mobility shift assays, whereas mutant M(C169S) did not (data not
shown). These results tentatively suggested that Cys154 and
Cys169 were important in maintaining activity, while
Cys55 was relatively benign. Furthermore, the catalytic
involvement by Cys154 remained a possibility, since mutant
M(C154S) maintained the ability to bind but not to modify. Although Cys
and Ser are structurally similar in the sense that they are both
nucleophilic, they possess significant differences in polarity. The
hydroxyl group on Ser is highly polar, while the sulfhydryl on Cys is
relatively nonpolar. Assuming Cys occupies a position in a hydrophobic
environment, the introduction of a polar residue like Ser could
significantly perturb the active enzyme structure. In order to address
this possibility, a second set of mutants was generated that replaced Cys by the nonpolar residue Ala. Since Ser substitution at positions 154 and 169 had the greatest effect on activity, these positions were
individually changed to Ala. In contrast to the Ser mutants, however,
both Ala mutants, M(C154A) and M(C169A), showed similar activity to
wild-type enzyme (Table I). This result suggests that the loss of
activity in the serine mutants M(C154S) and M(C169S) is not
mechanistically based but is more likely due to a perturbation of the
active enzyme structure by serine substitution. To further confirm
these results, a triple mutant was generated in which all three
cysteine residues in positions 55, 154, and 169 were changed to
alanine. Again, similar findings were observed (Table I). The triple
mutant, M(AAA), possessed high levels of activity, and its kinetic
parameters were compared with those of wild-type enzyme (Fig. 4). Both
wild-type and triple mutant enzymes exhibited comparable kinetic
parameters in Km and Vmax. A
1.2-fold increase in Km and a 1.6-fold decrease in
Vmax were observed for the mutant enzyme as
compared with wild-type enzyme. This resulted in an overall 2-fold
decrease in the parameter,
Vmax/Km. Since this parameter
is a measure of enzyme efficiency, the Cys to Ala mutations resulted in
the generation of a relatively active but somewhat less efficient
enzyme in both binding and Vmax. More important,
however, is the magnitude of the overall change in Vmax/Km, which is relatively
small. This suggests that the wild-type and mutant enzymes proceed by a
common mechanism that does not involve a covalent cysteine
intermediate. To further substantiate the thiol independence on
catalysis,
activity was also examined in the presence of the thiol
modification reagent, pCMB (Table II). Analogous to wild-type PSUI, the
enzymatic activities of M(C154A) and M(C169A) were lost in the presence
of pCMB, but upon subsequent addition of DTT the activities were
restored. The triple mutant, M(AAA), however, which lacks cysteine
residues, was not significantly affected by the addition of pCMB. Only
a slight diminution in activity was observed. This result verifies that
the activity measured for mutant M(AAA) was solely due to mutant enzyme
and not the presence of endogenous wild-type enzyme, since wild-type
protein is virtually inactive under these conditions. From these
experiments, it is clear that cysteine residues are not necessary
for maintaining catalysis during
formation. Additionally, Cys154 and Cys169 in the wild-type enzyme may
occupy a hydrophobic position within the protein fold that is sensitive
to substitution by a polar substituent and/or chemical
modification.
The formation of is not a simple process; it involves a multistep
mechanism. PSUI has to first bind the tRNA, next cleave the uracil base
from ribose, and then rotate and ligate it back on the ribose of the
tRNA. All of these events occur in a site-specific manner on the tRNA.
Chemically, the cleavage step in the formation of
is catalytically
similar to the cleavage event seen in uracil-DNA glycosylases that
catalyze the cleavage of uracil residues during DNA repair (30). In
principle, mutants that lack cysteine residues could facilitate the
initial step in the U to
rearrangement, which involves cleavage of
the carbon-nitrogen glycosyl bond. If a covalent cysteine intermediate
was necessary for retaining uracil after cleavage from ribose, then the
absence of a catalytic cysteine residue could result in the
site-specific formation of an abasic site. Since the tritium release
assay involves the absorption of substrates on charcoal, this assay is
able to distinguish between the release of free
[5-3H]uracil (resulting from glycosylase activity) and
3H+ (resulting from
formation). This was
verified using [5-3H]uracil as a control. In all
experiments with Cys to Ala mutant enzymes, no evidence of the
formation of abasic sites could be detected. Finally, further evidence
was provided using aniline-facilitated chemical cleavage of modified
tRNA (25). For tRNAs modified by wild-type and mutant enzymes, no
cleavage of the tRNA was observed when incubated under acidic
conditions followed by treatment with aniline (data not shown). These
results indicate that no abasic sites were generated in tRNA after
modification with mutant PSUI enzymes, thus validating the mechanistic
interpretation of the Ala mutants.
In conclusion, mechanistic pathways are never proven; rather, the
elimination of plausible routes serves to support the hypothesis. The
catalytic formation of residues by PSUI does not involve covalent
cysteine-derived intermediates as previously thought. This may be a
general feature of
synthases. Furthermore, mutational studies
presented here demonstrate that enzyme activity is still maintained
even in the absence of Cys residues within the protein. Mechanistically, the formation of
appears to resemble more closely the action of a uracil glycosylase than it does a methyltransferase. The active site of this enzyme is no doubt guarded from releasing uracil after its initial cleavage from ribose to allow for rotation and
reattachment at C5. Current efforts are underway involving
crystallization trials of PSUI bound to its tRNA substrate in order to
elucidate the mechanistic details of this intriguing rearrangement.