(Received for publication, October 11, 1995; and in revised form, December 6, 1995)
From the
Bovine pancreatic ribonuclease A catalyzes the depolymerization
of RNA. There is much evidence that several subsites, in addition to
the main catalytic site, are involved in the formation of the
enzyme-substrate complex. This work analyzes the pattern of
oligonucleotide formation by ribonuclease A using poly(C) as substrate.
The poly(C) cleavage shows that the enzyme does not act in a random
fashion but rather prefers the binding and cleavage of the longer
substrate molecules and that the phosphodiester bond broken should be
6-7 residues apart from the end of the chain to be preferentially
cleaved by ribonuclease A. The results demonstrate the model of the
cleavage of an RNA chain based on the cooperative binding between the
multisubsite binding structure of ribonuclease A and the phosphates of
the polynucleotide (Parés, X.,
Nogués, M. V., de Llorens, R., and Cuchillo, C.
M.(1991) in Essays in Biochemistry (Tipton, K. F., ed) Vol.
26, pp. 89-103, Portland Press Ltd., London). The contribution to
the enzymatic process of the non-catalytic phosphate-binding subsite
(p) adjacent to the catalytic center has been analyzed in
p
chemically modified ribonuclease A or by means of
site-directed mutagenesis. In both cases deletion of p
abolishes the endonuclease activity of ribonuclease A, which is
substituted by an exonuclease activity. All these results support the
role of the multisubsite structure of the enzyme in the endonuclease
activity and in the catalytic mechanism.
Bovine pancreatic ribonuclease A (RNase A) ()(EC
3.1.27.5) is a well known enzyme whose main physical, chemical, and
enzymatic properties have been the subject of extensive reviews
(Richards and Wyckoff, 1971; Blackburn and Moore, 1982; Eftink and
Biltonen, 1987). RNase A is an endonuclease that cleaves
3`,5`-phosphodiester linkages of single-stranded RNA when the base of
the nucleotide in the 3` position is a pyrimidine. The use of well
defined low molecular mass substrates such as pyrimidine 2`,3`-cyclic
mononucleotides and dinucleoside monophosphates provided most of the
kinetic data on which all mechanistic studies on RNase A have relied.
Only a few kinetic studies have been carried out with longer
oligonucleotides and homopolynucleotides such as poly(U) (Irie et
al., 1984a, 1984b), but no detailed studies with RNA as substrate
have been carried out. The reasons for this are (i) the difficulties in
the kinetic analysis derived from the complex structural features of
the RNA molecule, (ii) the difficulty of monitoring a very fast
reaction in a reliable fashion, and (iii) the spectrophotometric
methods used can only give an average measure of all the species
produced during the reaction, without giving any idea about either the
size distribution of the products or the characteristics of the bond
broken. The use of HPLC techniques has circumvented some of the
problems concerning the analysis of the products of the reaction. Using
an anion exchange column it was possible to measure the products of the
reaction at high concentrations of the low molecular mass substrate
cytidine 2`,3`-cyclic phosphate (C>p). This method allowed the
analysis of the partition of the reaction between the synthesis of the
dinucleotide CpC>p and the hydrolysis to 3`-CMP (Guasch et
al., 1989; Cuchillo et al., 1991) and the separation
between transphosphorylation and hydrolysis reactions using CpC as
substrate (Cuchillo et al., 1993). In addition to the
catalytic center, several phosphate-binding subsites that recognize the
negatively charged phosphates of RNA have been described
(Parés et al., 1980;
Parés et al., 1991; Fontecilla-Camps et al., 1994; Nogués et al.,
1995) (Fig. 1). A non-catalytic phosphate-binding subsite
(p
) adjacent to the catalytic center (p
) was
postulated from the chemical modification of RNase A with the
halogenated nucleotide 6-chloropurine-9-
-D-ribofuranosyl
5`-monophosphate (cl
RMP) (Parés et al., 1980). The reaction yielded a major derivative
(derivative II) with the nucleotide label attached to the
-amino
group. Different studies suggested that the phosphate group was bound
in a specific phosphate-binding subsite, p
, and that Lys-7
and Arg-10 were involved in that subsite (Arús et al., 1981; Irie et al., 1986; de Llorens et
al., 1989; Richardson et al., 1990). The structure of
derivative II was recently solved by x-ray crystallography
(Boquéet al., 1994), and the structure
found is in accordance with the location of p
,
B
, and R
(
)subsites at the
N-terminal region of the protein. By means of kinetic analysis of RNase
A derivatives obtained by site-directed mutagenesis, an indirect role
for the p
subsite in the enzyme's catalytic mechanism
was demonstrated (Boix et al., 1994). In the present work the
pattern of oligonucleotide formation by RNase A using poly(C) as
substrate was analyzed by means of reversed-phase HPLC, and the
contribution of the binding subsite p
to the product
distribution was studied in both native RNase A and in
p
-modified molecules. The results indicate that in the
poly(C) cleavage RNase A does not act in a random fashion but rather
prefers the binding and cleavage of the longer substrate molecules. In
addition the phosphodiester bond broken should be 6-7 nucleotides
apart from at least one of the ends to be preferentially cleaved by
RNase A. Finally, deletion of p
abolishes the endonuclease
activity of RNase A, which is substituted by an exonuclease activity.
All these facts can be explained in terms of the multisubsite structure
of the enzyme.
Figure 1:
Schematic diagram of the RNase A active
site and adjacent binding subsites. B, R, and p refer to the binding
subsites for base, ribose, and phosphate, respectively. B is specific for pyrimidines and B
``prefers'' purines. The phosphate group of the
phosphodiester bond hydrolyzed by the enzyme binds to p
.
Amino acids located near each site are indicated
(Parés et al.,
1991).
A similar process was used to analyze the cleavage of poly(C) by derivative II and K7Q and K7Q plus R10Q RNase A mutants. In these cases the initial enzyme concentrations were 50, 35, and 62 nM, respectively.
Figure 2:
Analysis by reversed-phase HPLC column
(Nova-Pak C column) of products obtained from poly(C)
digestion by RNase A at time intervals from 0 to 45 min. See
``Methods'' for digestion and separation conditions. Note
that in each chromatogram the best scale on the ordinate is
used.
Fig. 2also shows the elution profile by reversed-phase HPLC
of the (Cp)C>p oligonucleotides obtained from the
poly(C) digestion with a low concentration of RNase A at different
times within the range 0-45 min. The elution position of the
small oligonucleotides obtained from poly(C) digestion by RNase A was
deduced from the pattern obtained after the reaction mixture was
incubated for a long time (100 min) when no high molecular mass poly(C)
was left (Fig. 3). The separation pattern found by McFarland and
Borer(1979) for the chemical hydrolysis of poly(C) was also taken into
account for the identification of the different peaks. Previous studies
about the degradation of poly(C), poly(U), and poly(A) by RNase A had
shown that the oligoribonucleotidic acids have a general structure of
(Cp)
C>p containing a 2`,3`-cyclic phosphate terminus,
except for the fragment arising from the 3` terminus of the initial
molecules of substrate (Imura et al., 1965; Irie et
al., 1984b). The two mononucleotide products 3`-CMP and C>p
elute very near the injection peak and thus they cannot be directly
measured. The smallest structure that can be clearly separated is the
dinucleotide CpC>p with an elution time of around 4 min. Oligomers
of increasing size elute sequentially as a function of the amount of
organic phase in the eluent.
Figure 3:
Elution profile on a reversed-phase HPLC
column (Nova-Pak C column) of oligocytidylic acids
(Cp)
C>p (n = 1-5) from poly(C)
digestion by RNase A during 100 min. See ``Methods'' for
digestion and separation conditions.
The analysis of the oligonucleotide size distribution (Fig. 2, 4, and 5) shows that under the conditions used only polynucleotide fragments are formed during the early stages of incubation. However, shortly thereafter (5 min) a clear trend toward the formation of oligonucleotides with a size of about 6-7 residues is observed. As expected, at the end of the process there is a clear increase in the number of small size oligonucleotides. These results suggest that the enzyme prefers binding and cleavage of long substrates and that to be preferentially cleaved by RNase A the phosphodiester bond has to be some six-seven nucleotides apart from at least one of the ends of the molecule.
The rate of appearance of
mononucleotides C>p and 3`-CMP was followed by rechromatography on
an anion exchange HPLC column (Nucleosil 10SB) of the fraction eluted
during the first 2 min of chromatography of poly(C) digestion products
on the Nova-Pak C column (Table 1). The formation of
mononucleotides is a very slow process. For example, after 45 min of
poly(C) digestion when no poly(C) is left but oligonucleotides are
present in the medium as RNase A substrates (Fig. 2), the
absorbance area at 260 nm of mononucleotide fraction accounts for only
14% of the total, 92% of which is C>p and the remaining 8% of which
is 3`-CMP. In agreement with previous results on CpC digestion by RNase
A (Guasch et al., 1989; Cuchillo et al., 1993) there
is an accumulation of C>p in the reaction medium before its
transformation to 3`-CMP.
Figure 4:
Distribution of the fractions
corresponding to poly(C), (Cp)C>p, and CpC>p during
the digestion process of poly(C) by RNase A. Area percent of each
compound was obtained from the peak area integration of Fig. 2.
Note that the left scale corresponds to poly(C) (
) and
the right scale to (Cp)
C>p (
) and
CpC>p (
).
The hexanucleotide species (Cp)C>p behaves as a
transient intermediate, whereas CpC>p accumulates during the
observed reaction times. This product distribution suggested that RNase
A shows a clear preference for polynucleotide or oligonucleotide
substrates of high molecular mass rather than for low molecular mass
substrates. These results can be explained according to the
multisubsite structure of RNase A (Parés et
al., 1991; Nogués et al., 1995).
The formation of the RNA
RNase A complex is mainly driven by
interactions between the phosphate groups of the substrate and the
active site (p
) and the main phosphate-binding subsites
(p
and p
) of the enzyme (Fig. 1).
However, other electrostatic interactions between phosphate groups of
RNA and basic amino acid residues located at the surface of the protein
are also involved in leading to optimal catalytic efficiency. The total
occupancy of these binding subsites gives the best conformation for
activity, and the additional binding energy clearly favors the action
on the higher substrates.
For these reasons we chose
derivative II and K7Q and K7Q plus R10Q RNase A mutants to check the
role of the p phosphate-binding subsite in the poly(C)
cleavage pattern by RNase A. In all three derivatives substrate binding
in the p
subsite is greatly decreased due to either the
occupancy of this subsite by the phosphate group of the marker
nucleotide (derivative II) or to the partial (K7Q RNase A mutant) or
total absence (K7Q plus R10Q RNase A mutant) of positive charges
necessary for the enzyme-substrate electrostatic interactions in this
region.
Fig. 5clearly shows that differences in
oligonucleotide formation pattern take place as a consequence of a
non-functional p binding subsite. Due to the lower enzyme
activity of RNase A mutants with respect to the native enzyme (Boix et al., 1994) the comparison has been established between
digestion products containing the same percentage of undigested poly(C)
fraction that is at equivalent stages of the digestion process. In
contrast with the native enzyme, the cleavage of poly(C) by the RNases
modified at p
shows a pattern in which, from the initial
reaction time, the major product formed corresponds to the
mononucleotide fraction and other small products, without accumulation
of the hexanucleotide. This behavior is more apparent in both the
derivative II and the K7Q plus R10Q with a totally non-functional
p
binding site than with K7Q in which there is still a
positive charge near p
. These findings indicate that the
modified RNase cleaves the substrate in an exonucleolytic fashion in
contrast with native RNase A, which acts preferentially as an
endonuclease.
Figure 5:
Comparison of
(Cp)C>p (n = 0-8)
formation from poly(C) cleavage by RNase A (
) and RNase
A-modified forms (derivative II, K7Q plus R10Q RNase A mutant, and K7Q
RNase A mutant as marked on the panels)
([&cjs2113;]). Area percent in each case has been determined
from the area of the corresponding peak eluted from the Nova-Pak
C
column. Due to the different enzyme activities
comparisons have been established using as reference the same
undigested poly(C) fraction. Note that in each graphic the best scale
on the ordinate is used.
The results shown in Fig. 2indicate that the
breakdown of poly(C) catalyzed by RNase A is not a random process even
though all internucleotide bonds in the substrate are susceptible to
attack by the enzyme. Instead, it can be considered as taking place
roughly in consecutive steps; during the early part of the reaction
longer fragments are expected for a random endolytic reaction, but as
the reaction proceeds there is a significant accumulation of
oligocytidylic acids of 6-7 residues, which in the final stages
of the reaction are transformed into the mononucleotide C>p and
eventually into 3`-CMP ( Fig. 4and Table 1). These results
suggest that the enzyme prefers binding and cleavage of long substrates
and that the phosphodiester bond broken should be some 6-7
residues apart from the end of the chain to be preferentially cleaved
by RNase A. It should be noted that although this treatment is
indicative of the behavior of RNase A on poly(C) it cannot be used to
calculate accurately the kinetic parameters. This is because (i)
although the hexanucleotide product has been taken as the main
intermediate one should consider a broader population of products and
(ii) the same enzyme concentration has been used for the two
consecutive steps, i.e. the initial concentration, but the
relative concentration of enzyme in each step is smaller as they are
catalyzed by the same enzyme which, therefore, is partitioned between
the two steps. An accurate knowledge of the k/K
for each substrate
would allow knowledge of the proportion of enzyme acting in each
reaction.
These results are supported by kinetic studies that
suggest the contribution of phosphate-binding subsites to the
catalysis. Steady-state kinetic studies of RNase A with
oligonucleotides as substrates indicate that the k values increase with the substrate size, the k
value for UpApA and UpApG being 3-5 times higher than those
of UpA (Irie et al., 1984a). The use of oligouridylic acids
has shown similar results (Irie et al., 1984b). From these
findings it is demonstrated that the p
, p
, and
p
binding sites play an important role in catalysis.
Moreover, when poly(U) is used as substrate the V
value is 3-20 times higher than the values with
oligouridylic acids depending on the assay conditions (Irie et
al., 1984b). These kinetic results suggest that additional binding
subsites must contribute to the catalytic efficiency. Crystallographic
analysis of complexes between the protein and the deoxyadenylic acid
tetramer supports the existence of multiple subsites in RNase A. A
virtual DNA strand composed of 12 nucleotides traces out a nearly
continuous path. The binding between protein and nucleic acid takes
place through salt bridges between phosphate groups and nine charged
side chains (McPherson et al., 1986).
Our results support the model of the cleavage of an RNA chain based on cooperative binding between the multisubsite structure of RNase A and the phosphates of the polynucleotide as proposed by Parés et al.(1991) (Fig. 6). This model proposes that the strong binding between RNA and RNase A takes place primarily through electrostatic interactions between the phosphate groups of the substrate and basic amino acid residues located on the surface of the protein. After the cleavage of the phosphodiester bond at the active site the cooperative binding is weakened in the fragments, and the displacement by a new long chain of RNA occurs. Subsequently, shorter fragments will be progressively cleaved in the order of maximum to minimum occupancy of the RNase A subsites. Finally, the hydrolytic step takes place.
Figure 6: Model of the cleavage of an RNA chain by RNase A that explains the preference of the enzyme for long polynucleotide substrates. The model is based on the cooperative binding between the multiple protein subsites and the phosphates of the polynucleotide. Step 1, a long RNA chain binds to RNase A. Step 2, cleavage occurs in the active site resulting in the formation of two shorter oligonucleotide fragments, one of them ending with a 2`,3`-cyclic phosphate (2`,3`c). Step 3, the cooperative binding is weakened in the oligonucleotide fragments and this favors their replacement by a longer chain. In subsequent reactions the shorter fragments will also be broken, and eventually the hydrolytic step (formation of a 3`-phosphate from the 2`,3`-cyclic phosphodiesters) occurs when most of the substrate has already been cleaved by transphosphorylation. Adapted from Parés et al.(1991).
The contribution of phosphate-binding subsites to the
enzymatic process has been analyzed in p chemically
modified RNase A (derivative II) or by means of site-directed
mutagenesis (K7Q and K7Q plus R10Q mutants). In both cases the product
distribution pattern from poly(C) cleavage is altered (Fig. 5),
and the significant increase in the mononucleotide fraction with
respect to the production of oligonucleotides indicates that in these
modified RNase A forms an exonuclease activity is favored. The RNase
specificity has also been altered by site-directed mutagenesis of amino
acid residues of B
, the pyrimidine binding site
(delCardayré and Raines, 1994); the replacement
of Thr-45 by amino acids with smaller side chains (i.e. Ala or
Gly) increases the efficiency for poly(A) transphosphorylation up to
the level of poly(C), and at the same time a processive poly(A)
cleavage is seen in contrast with the distributive cleavage
characteristic of wild-type RNase A. Thus, these substitutions not only
modify the specificity of substrate binding, but they also alter the
cleavage pattern. In addition, an indirect role for the amino acid
residues of the p
binding subsite in the RNase A catalytic
process has been demonstrated, since the catalytic efficiency is
strongly affected not only in the case of an RNA substrate, which
occupies all the binding subsites, but also in the case of the C>p
substrate, which only interacts in the catalytic center
(B
R
p
) (Boix et al., 1994).
In conclusion, the best catalytic efficiency of RNase A is produced
with long substrates as a consequence of the occupation of all the
phosphate-binding sites. The p
phosphate-binding subsite
also plays an important role in the catalytic mechanism and contributes
significantly to the endonuclease activity of RNase A.