RNA cleavage without hydrolysis. Splitting the catalytic activities of binase with Asn101 and Thr101 mutations

A L  Okorokov; K I  Panov; W A  Offen; V G  Mukhortov; A A  Antson; M Y  Karpeisky; A J  Wilkinson; G G  Dodson

RNA cleavage without hydrolysis. Splitting the catalytic activities of binase with Asn101 and Thr101 mutations

A L Okorokov, K I Panov, W A Offen, V G Mukhortov, A A Antson, M Y Karpeisky, A J Wilkinson, G G Dodson

Chemistry

Research output: Contribution to journal › Article › peer-review

Abstract

Members of the microbial guanyl-specific ribonuclease family catalyse the endonucleolytic cleavage of single-stranded RNA in a two-step reaction involving transesterification to form a 2',3'-cyclic phosphate and its subsequent hydrolysis to yield the respective 3'-phosphate, The extracellular ribonuclease from Bacillus intermedius (binase, RNase Ri) shares a common mechanism for RNA hydrolysis with mammalian RNases, Two catalytic residues in the active site of binase, Glu72 and His101, are thought to be involved in general acid-general base catalysis of RNA cleavage, Using site-directed mutagenesis, binase mutants were produced containing amino acid substitutions H101N and H101T and their catalytic properties towards RNA, poly(I), poly(A), GPC and guanosine 2',3'-cyclic phosphate (cGMP) substrates were studied, The engineered mutant proteins are active in the transesterification step which produces the 2',3'-cyclic phosphate species but they have lost the ability to catalyse hydrolysis of the cyclic phosphate to give the 3' monophosphate product.

Original language	English
Pages (from-to)	273-278
Number of pages	6
Journal	PROTEIN ENGINEERING DESIGN
Volume	10
Issue number	3
Publication status	Published - Mar 1997

Keywords

active-site mutants
binase
ribonuclease
site-directed mutagenesis
MICROBIAL RIBONUCLEASES
3-DIMENSIONAL STRUCTURE
BARNASE
RESOLUTION
EXPRESSION
SPECIFICITY
REFINEMENT
INHIBITOR
MECHANISM
SUBSITES

Cite this

@article{3346ec7158d04bd7b580abd12b0d2ab2,

title = "RNA cleavage without hydrolysis. Splitting the catalytic activities of binase with Asn101 and Thr101 mutations",

abstract = "Members of the microbial guanyl-specific ribonuclease family catalyse the endonucleolytic cleavage of single-stranded RNA in a two-step reaction involving transesterification to form a 2',3'-cyclic phosphate and its subsequent hydrolysis to yield the respective 3'-phosphate, The extracellular ribonuclease from Bacillus intermedius (binase, RNase Ri) shares a common mechanism for RNA hydrolysis with mammalian RNases, Two catalytic residues in the active site of binase, Glu72 and His101, are thought to be involved in general acid-general base catalysis of RNA cleavage, Using site-directed mutagenesis, binase mutants were produced containing amino acid substitutions H101N and H101T and their catalytic properties towards RNA, poly(I), poly(A), GPC and guanosine 2',3'-cyclic phosphate (cGMP) substrates were studied, The engineered mutant proteins are active in the transesterification step which produces the 2',3'-cyclic phosphate species but they have lost the ability to catalyse hydrolysis of the cyclic phosphate to give the 3' monophosphate product.",

keywords = "active-site mutants, binase, ribonuclease, site-directed mutagenesis, MICROBIAL RIBONUCLEASES, 3-DIMENSIONAL STRUCTURE, BARNASE, RESOLUTION, EXPRESSION, SPECIFICITY, REFINEMENT, INHIBITOR, MECHANISM, SUBSITES",

author = "Okorokov, {A L} and Panov, {K I} and Offen, {W A} and Mukhortov, {V G} and Antson, {A A} and Karpeisky, {M Y} and Wilkinson, {A J} and Dodson, {G G}",

year = "1997",

month = mar,

language = "English",

volume = "10",

pages = "273--278",

journal = "Protein Engineering, Design and Selection",

issn = "1741-0126",

publisher = "Oxford University Press",

number = "3",

}

TY - JOUR

T1 - RNA cleavage without hydrolysis. Splitting the catalytic activities of binase with Asn101 and Thr101 mutations

AU - Okorokov, A L

AU - Panov, K I

AU - Offen, W A

AU - Mukhortov, V G

AU - Antson, A A

AU - Karpeisky, M Y

AU - Wilkinson, A J

AU - Dodson, G G

PY - 1997/3

Y1 - 1997/3

N2 - Members of the microbial guanyl-specific ribonuclease family catalyse the endonucleolytic cleavage of single-stranded RNA in a two-step reaction involving transesterification to form a 2',3'-cyclic phosphate and its subsequent hydrolysis to yield the respective 3'-phosphate, The extracellular ribonuclease from Bacillus intermedius (binase, RNase Ri) shares a common mechanism for RNA hydrolysis with mammalian RNases, Two catalytic residues in the active site of binase, Glu72 and His101, are thought to be involved in general acid-general base catalysis of RNA cleavage, Using site-directed mutagenesis, binase mutants were produced containing amino acid substitutions H101N and H101T and their catalytic properties towards RNA, poly(I), poly(A), GPC and guanosine 2',3'-cyclic phosphate (cGMP) substrates were studied, The engineered mutant proteins are active in the transesterification step which produces the 2',3'-cyclic phosphate species but they have lost the ability to catalyse hydrolysis of the cyclic phosphate to give the 3' monophosphate product.

AB - Members of the microbial guanyl-specific ribonuclease family catalyse the endonucleolytic cleavage of single-stranded RNA in a two-step reaction involving transesterification to form a 2',3'-cyclic phosphate and its subsequent hydrolysis to yield the respective 3'-phosphate, The extracellular ribonuclease from Bacillus intermedius (binase, RNase Ri) shares a common mechanism for RNA hydrolysis with mammalian RNases, Two catalytic residues in the active site of binase, Glu72 and His101, are thought to be involved in general acid-general base catalysis of RNA cleavage, Using site-directed mutagenesis, binase mutants were produced containing amino acid substitutions H101N and H101T and their catalytic properties towards RNA, poly(I), poly(A), GPC and guanosine 2',3'-cyclic phosphate (cGMP) substrates were studied, The engineered mutant proteins are active in the transesterification step which produces the 2',3'-cyclic phosphate species but they have lost the ability to catalyse hydrolysis of the cyclic phosphate to give the 3' monophosphate product.

KW - active-site mutants

KW - binase

KW - ribonuclease

KW - site-directed mutagenesis

KW - MICROBIAL RIBONUCLEASES

KW - 3-DIMENSIONAL STRUCTURE

KW - BARNASE

KW - RESOLUTION

KW - EXPRESSION

KW - SPECIFICITY

KW - REFINEMENT

KW - INHIBITOR

KW - MECHANISM

KW - SUBSITES

M3 - Article

VL - 10

SP - 273

EP - 278

JO - Protein Engineering, Design and Selection

JF - Protein Engineering, Design and Selection

SN - 1741-0126

IS - 3

ER -