Abstract:Ribonucleases have been found to have subsites that confer large rate enhancements but do not contribute to substrate binding. In this study, we present a kinetic model that formally explains how subsite binding energy is converted into chemical activation energy. The proposed mechanism takes into account a primary specificity site and a subsite, both of which must be occupied for chemical turnover. An unstable reaction intermediate is formed upon binding of the polymeric substrate monomers at the correspondin… Show more
“…A simple kinetic model considering sequential binding at the primary site and the subsite ( Fig. 6) accounts for the steady state behavior of RNase T, [54]. If we assume equilibrium between all species, the apparent steady-state kinetic parameters for this model are as follows (see [54] for the complete steady-state treatment).…”
Section: Nature Of the Subsite Interactionsmentioning
confidence: 99%
“…6) accounts for the steady state behavior of RNase T, [54]. If we assume equilibrium between all species, the apparent steady-state kinetic parameters for this model are as follows (see [54] for the complete steady-state treatment). The observation that mutations at the subsite affect turnover rather than binding 1471 indicates (Eqns 1 and 2 ) that an unstable reaction intermediate (ES) is formed upon binding of the substrate monomers at the corresponding subsites (K,,,, > 1 or b A G,,,,, < d GEs ; Fig.…”
Section: Nature Of the Subsite Interactionsmentioning
During the last decade, protein engineering has been used to identify the residues that contribute to the ribonulease-TI-catalyzed transesterification. His40, Glu58 and His92 accelerate the associative nucleophilic displacement at the phosphate atom by the entering 2'-oxygen downstream guanosines in a highly cooperative manner. Glu58, assisted by the protonated His40 imidazole, abstracts a proton from the %'-oxygen, while His92 protonates the leaving group. Tyr38, Arg77 and PhelOO further stabilize the transition state of the reaction. A functionally independent subsite, including Am36 and Asn98, contributes to chemical turnover by aligning the substrate relative to the catalytic side chains upon binding of the leaving group. An invariant structural motive, involving residues 42-46, renders ribonuclease T I guanine specific through a series of intermolar hydrogen bonds. Tyr42 contributes significantly to guanine binding through a parallel face-to-face stacking interaction. Tyr45, often referred to as the lid of the guanine-binding site, does not contribute to the binding of the base.Keywords: ribonuclease T, ; protein engineering; catalytic mechanism ; functional cooperativity ; substrate specificity ; subsite interaction ; nucleotide binding ; double-mutant cycle.Ribonuclease T , (RNase TI) of the slime mould Aspergillus oryzae [ 1-41 is the best known representative of a large family of homologues microbial ribonucleases with members in the prokaryotic and the eukaryotic world [5, 61. These ribonucleases span the greatest evolutionary divide of all known protein families. RNase TI, a guanosine-specific ribonuclease, was first isolated by Sato and Egami [ I ] from an enzyme extract (Takadiastase) of the culture medium of A. oryzae used in the malting process of sake brewing. The enzyme consists of a single polypeptide chain of 104 residues of known sequence [7, 81 and contains two disulfide bridges. RNase T1 is a stable acidic protein; it is fairly resistant to heat, acids, and bases. A large number of crystal structures of RNase T, are available in different liganded states [9-221. The general fold (Fig. 1) consists of a 4.5-turn a helix and two antiparallel p sheets, connected through a series of wide loops [9, 101. The residues implicated in catalysis are anchored in the major p sheet, while guanosine specificity is defined by residues in loop regions.RNase T, cleaves the P-05' ester bond of GpN sequences of single-stranded RNA by a transphosphorylation reaction (Fig. 2), yielding a 2',3'-cyclophosphate [I, 23, 241. In a second, separate step, this cyclic product may be hydrolyzed to yield 3'-guanylic acid [25 -271. The catalyzed transesterification reaction consists of an associative nucleophilic displacement at the phosphorus atom of the 5' leaving group by the entering 2'-oxygen atom. The enzyme is thought to follow a concerted inline mechanism with a trigonal bipyramidal transition state, implying a base and an acid located on either side of the scissile Correspondence to J. Steyaert, Dienst Ultrastruktu...
“…A simple kinetic model considering sequential binding at the primary site and the subsite ( Fig. 6) accounts for the steady state behavior of RNase T, [54]. If we assume equilibrium between all species, the apparent steady-state kinetic parameters for this model are as follows (see [54] for the complete steady-state treatment).…”
Section: Nature Of the Subsite Interactionsmentioning
confidence: 99%
“…6) accounts for the steady state behavior of RNase T, [54]. If we assume equilibrium between all species, the apparent steady-state kinetic parameters for this model are as follows (see [54] for the complete steady-state treatment). The observation that mutations at the subsite affect turnover rather than binding 1471 indicates (Eqns 1 and 2 ) that an unstable reaction intermediate (ES) is formed upon binding of the substrate monomers at the corresponding subsites (K,,,, > 1 or b A G,,,,, < d GEs ; Fig.…”
Section: Nature Of the Subsite Interactionsmentioning
During the last decade, protein engineering has been used to identify the residues that contribute to the ribonulease-TI-catalyzed transesterification. His40, Glu58 and His92 accelerate the associative nucleophilic displacement at the phosphate atom by the entering 2'-oxygen downstream guanosines in a highly cooperative manner. Glu58, assisted by the protonated His40 imidazole, abstracts a proton from the %'-oxygen, while His92 protonates the leaving group. Tyr38, Arg77 and PhelOO further stabilize the transition state of the reaction. A functionally independent subsite, including Am36 and Asn98, contributes to chemical turnover by aligning the substrate relative to the catalytic side chains upon binding of the leaving group. An invariant structural motive, involving residues 42-46, renders ribonuclease T I guanine specific through a series of intermolar hydrogen bonds. Tyr42 contributes significantly to guanine binding through a parallel face-to-face stacking interaction. Tyr45, often referred to as the lid of the guanine-binding site, does not contribute to the binding of the base.Keywords: ribonuclease T, ; protein engineering; catalytic mechanism ; functional cooperativity ; substrate specificity ; subsite interaction ; nucleotide binding ; double-mutant cycle.Ribonuclease T , (RNase TI) of the slime mould Aspergillus oryzae [ 1-41 is the best known representative of a large family of homologues microbial ribonucleases with members in the prokaryotic and the eukaryotic world [5, 61. These ribonucleases span the greatest evolutionary divide of all known protein families. RNase TI, a guanosine-specific ribonuclease, was first isolated by Sato and Egami [ I ] from an enzyme extract (Takadiastase) of the culture medium of A. oryzae used in the malting process of sake brewing. The enzyme consists of a single polypeptide chain of 104 residues of known sequence [7, 81 and contains two disulfide bridges. RNase T1 is a stable acidic protein; it is fairly resistant to heat, acids, and bases. A large number of crystal structures of RNase T, are available in different liganded states [9-221. The general fold (Fig. 1) consists of a 4.5-turn a helix and two antiparallel p sheets, connected through a series of wide loops [9, 101. The residues implicated in catalysis are anchored in the major p sheet, while guanosine specificity is defined by residues in loop regions.RNase T, cleaves the P-05' ester bond of GpN sequences of single-stranded RNA by a transphosphorylation reaction (Fig. 2), yielding a 2',3'-cyclophosphate [I, 23, 241. In a second, separate step, this cyclic product may be hydrolyzed to yield 3'-guanylic acid [25 -271. The catalyzed transesterification reaction consists of an associative nucleophilic displacement at the phosphorus atom of the 5' leaving group by the entering 2'-oxygen atom. The enzyme is thought to follow a concerted inline mechanism with a trigonal bipyramidal transition state, implying a base and an acid located on either side of the scissile Correspondence to J. Steyaert, Dienst Ultrastruktu...
“…(B) Ribonuclease A catalyzes transesterification of simple primary alkanols with 2‘,3‘-cyclic nucleoside phosphates, at a much reduced rate as compared to RNA cleavage ,, and by the fact that the cyclic phosphate is the physiological end-product of the RNA cleavage. , The low rate of the cleavage of 2‘,3‘-cyclic phosphate, mostly due to reduction in the k cat , results from the absence of a part of the substrate structure needed to bind to the p2 site, to achieve full activity. , In the case of PI-PLC the analogous rate reduction is brought about by the removal of the hydrophobic chains from the substrate. 4 …”
Section: Discussionmentioning
confidence: 99%
“…40,52 The low rate of the cleavage of 2′,3′-cyclic phosphate, mostly due to reduction in the k cat , results from the absence of a part of the substrate structure needed to bind to the p2 site, to achieve full activity. 53,54 In the case of PI-PLC the analogous rate reduction is brought about by the removal of the hydrophobic chains from the substrate.…”
Transesterification of primary alcohols with inositol 1,2-cyclic
phosphate (IcP) in the presence of
phosphatidylinositol-specific phospholipase C (PI-PLC) resulted in the
formation of O-alkyl inositol 1-phosphates.
The starting IcP was obtained in a single step by PI-PLC catalyzed
cleavage of phosphatidylinositol from the soybean
phospholipid. The transesterification reaction was performed with
a series of 20 structurally diverse hydroxyl
compounds, ranging in the structural complexity from methanol to the
serine-containing Ser-Tyr-Ser-Met tetrapeptide,
to give the corresponding phosphodiesters with 20−80% yields,
depending mainly on the solubility of alcohols in
water. The rates of transesterifications, and of the competing
hydrolysis of IcP to inositol 1-phosphate (IP), were
relatively insensitive to the alcohol structure. With polyhydroxyl
compounds such as glycerol and hexitols, the
enzyme displayed complete preference toward formation of the inositol
phosphate derivatives of the primary hydroxyl
groups. On the other hand, PI-PLC did not discriminate between
primary hydroxyl groups in different environments
and showed low stereoselectivity with racemic alcohols featuring a
chiral center at the β-position. The
O-alkyl
inositol phosphates formed were readily separable from the hydrolytic
product, IP, by the anion-exchange
chromatography, and were fully characterized by means of 1H
and 31P NMR and electrospray MS. Our
results
provide a new, simple, and efficient two-step synthetic route to
substituted O-alkyl inositol phosphates from
inexpensive
starting materials. The reported reaction was successfully applied
to synthesis of complex inositol phosphate
derivatives, as illustrated by inositol phosphoesters of mono- and
oligosaccharides, nucleosides and peptides. The
synthetic usefulness of this reaction, however, is not limited to the
examples shown. Because transesterification
activity of phospholipase C has not been reported before, its mechanism
is discussed in a broad context of mechanisms
of phosphoesterases.
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