Interaction of C225SR1 Mutant Subunit of Ribonucleotide Reductase with R2 and Nucleoside Diphosphates: Tales of a Suicidal Enzyme

S. S. Mao, T. P. Holler, Joseph M. Bollinger, Jr., G. X. Yu, M. I. Johnston, J. Stubbe

Research output: Contribution to journalArticle

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Abstract

Ribonucleotide reductase (RDPR) from Escherichia coli is composed of two subunits, Rl and R2, both of which are required to catalyze the conversion of nucleotides to deoxynucleotides. This reduction process is accompanied by oxidation of two cysteines within the active site to a disulfide. One of these putative active site cysteines, C225, has been mutated to a serine, and the properties of this mutant (C225SR1) have been investigated in detail. Incubation of C225SR1 and R2 with [3ʹ-3H, U-14C]UDP results in time-dependent inactivation of the enzyme! This inactivation is accompanied by production of 2.4 uracils, 3H2O, and 3H,14C-labeled protein with an absorbance change at 320 nm. There is an isotope effect (kH/K3H) on uracil production of 3.2. In addition, the tyrosyl radical on R2 is reduced. The observation of 3H20, indicative of 3ʹcarbon-hydrogen bond cleavage and loss of the tyrosyl radical, provides a direct test of our mechanistic hypothesis that cleavage of this bond occurs concomitantly with tyrosyl radical reduction. Incubation of [3ʹ-2H]UDP with C225SR1 and R2 resulted in a V and V/K isotope effect on loss of the radical of 2.0 and 2.0, respectively. These studies provide the first direct evidence for protein radical involvement in catalysis. Reduction of the tyrosyl radical on R2 is accompanied by a stoichiometric cleavage of the Rl polypeptide into two new polypeptides of 26 and 61 kDa. The 26-kDa polypeptide is the N-terminus of Rl, and hence cleavage of the polypeptide is occurring in the region of the mutation. The N-terminus of the 61-kDa polypeptide is blocked. The mutant RDPR is inactivated by three different mechanisms: loss of the tyrosyl radical on R2, alkylation of Rl by 2-methylene-3(2H)-furanone, and cleavage of Rl into two polypeptides. This sequence of events is also observed with the purine substrate ADP and requires the presence of the appropriate allosteric effector. Studies with a variety of single and double Rl mutants suggest that the cleavage reaction requires the presence of C462 and C439. Small amounts of dUDP (dADP) are also observed during the interaction of UDP (ADP) with C225SR1R2 and are attributed to the presence of contaminating heterodimer of wt-Rl and mutant. A single mutation of an active site cysteine to a serine has converted the normal substrates into mechanism-based inhibitors. A mechanism to accommodate these amazing results is presented.

Original languageEnglish (US)
Pages (from-to)9744-9751
Number of pages8
JournalBiochemistry
Volume31
Issue number40
DOIs
StatePublished - Feb 1 1992

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Ribonucleotide Reductases
Diphosphates
Nucleosides
Peptides
Uridine Diphosphate
Enzymes
Cysteine
Catalytic Domain
Uracil
Isotopes
Adenosine Diphosphate
Serine
Mutation
Alkylation
Substrates
Catalysis
Disulfides
Escherichia coli
ribonucleotide reductase R2 subunit
Hydrogen

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Mao, S. S. ; Holler, T. P. ; Bollinger, Jr., Joseph M. ; Yu, G. X. ; Johnston, M. I. ; Stubbe, J. / Interaction of C225SR1 Mutant Subunit of Ribonucleotide Reductase with R2 and Nucleoside Diphosphates : Tales of a Suicidal Enzyme. In: Biochemistry. 1992 ; Vol. 31, No. 40. pp. 9744-9751.
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abstract = "Ribonucleotide reductase (RDPR) from Escherichia coli is composed of two subunits, Rl and R2, both of which are required to catalyze the conversion of nucleotides to deoxynucleotides. This reduction process is accompanied by oxidation of two cysteines within the active site to a disulfide. One of these putative active site cysteines, C225, has been mutated to a serine, and the properties of this mutant (C225SR1) have been investigated in detail. Incubation of C225SR1 and R2 with [3ʹ-3H, U-14C]UDP results in time-dependent inactivation of the enzyme! This inactivation is accompanied by production of 2.4 uracils, 3H2O, and 3H,14C-labeled protein with an absorbance change at 320 nm. There is an isotope effect (kH/K3H) on uracil production of 3.2. In addition, the tyrosyl radical on R2 is reduced. The observation of 3H20, indicative of 3ʹcarbon-hydrogen bond cleavage and loss of the tyrosyl radical, provides a direct test of our mechanistic hypothesis that cleavage of this bond occurs concomitantly with tyrosyl radical reduction. Incubation of [3ʹ-2H]UDP with C225SR1 and R2 resulted in a V and V/K isotope effect on loss of the radical of 2.0 and 2.0, respectively. These studies provide the first direct evidence for protein radical involvement in catalysis. Reduction of the tyrosyl radical on R2 is accompanied by a stoichiometric cleavage of the Rl polypeptide into two new polypeptides of 26 and 61 kDa. The 26-kDa polypeptide is the N-terminus of Rl, and hence cleavage of the polypeptide is occurring in the region of the mutation. The N-terminus of the 61-kDa polypeptide is blocked. The mutant RDPR is inactivated by three different mechanisms: loss of the tyrosyl radical on R2, alkylation of Rl by 2-methylene-3(2H)-furanone, and cleavage of Rl into two polypeptides. This sequence of events is also observed with the purine substrate ADP and requires the presence of the appropriate allosteric effector. Studies with a variety of single and double Rl mutants suggest that the cleavage reaction requires the presence of C462 and C439. Small amounts of dUDP (dADP) are also observed during the interaction of UDP (ADP) with C225SR1R2 and are attributed to the presence of contaminating heterodimer of wt-Rl and mutant. A single mutation of an active site cysteine to a serine has converted the normal substrates into mechanism-based inhibitors. A mechanism to accommodate these amazing results is presented.",
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Interaction of C225SR1 Mutant Subunit of Ribonucleotide Reductase with R2 and Nucleoside Diphosphates : Tales of a Suicidal Enzyme. / Mao, S. S.; Holler, T. P.; Bollinger, Jr., Joseph M.; Yu, G. X.; Johnston, M. I.; Stubbe, J.

In: Biochemistry, Vol. 31, No. 40, 01.02.1992, p. 9744-9751.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Interaction of C225SR1 Mutant Subunit of Ribonucleotide Reductase with R2 and Nucleoside Diphosphates

T2 - Tales of a Suicidal Enzyme

AU - Mao, S. S.

AU - Holler, T. P.

AU - Bollinger, Jr., Joseph M.

AU - Yu, G. X.

AU - Johnston, M. I.

AU - Stubbe, J.

PY - 1992/2/1

Y1 - 1992/2/1

N2 - Ribonucleotide reductase (RDPR) from Escherichia coli is composed of two subunits, Rl and R2, both of which are required to catalyze the conversion of nucleotides to deoxynucleotides. This reduction process is accompanied by oxidation of two cysteines within the active site to a disulfide. One of these putative active site cysteines, C225, has been mutated to a serine, and the properties of this mutant (C225SR1) have been investigated in detail. Incubation of C225SR1 and R2 with [3ʹ-3H, U-14C]UDP results in time-dependent inactivation of the enzyme! This inactivation is accompanied by production of 2.4 uracils, 3H2O, and 3H,14C-labeled protein with an absorbance change at 320 nm. There is an isotope effect (kH/K3H) on uracil production of 3.2. In addition, the tyrosyl radical on R2 is reduced. The observation of 3H20, indicative of 3ʹcarbon-hydrogen bond cleavage and loss of the tyrosyl radical, provides a direct test of our mechanistic hypothesis that cleavage of this bond occurs concomitantly with tyrosyl radical reduction. Incubation of [3ʹ-2H]UDP with C225SR1 and R2 resulted in a V and V/K isotope effect on loss of the radical of 2.0 and 2.0, respectively. These studies provide the first direct evidence for protein radical involvement in catalysis. Reduction of the tyrosyl radical on R2 is accompanied by a stoichiometric cleavage of the Rl polypeptide into two new polypeptides of 26 and 61 kDa. The 26-kDa polypeptide is the N-terminus of Rl, and hence cleavage of the polypeptide is occurring in the region of the mutation. The N-terminus of the 61-kDa polypeptide is blocked. The mutant RDPR is inactivated by three different mechanisms: loss of the tyrosyl radical on R2, alkylation of Rl by 2-methylene-3(2H)-furanone, and cleavage of Rl into two polypeptides. This sequence of events is also observed with the purine substrate ADP and requires the presence of the appropriate allosteric effector. Studies with a variety of single and double Rl mutants suggest that the cleavage reaction requires the presence of C462 and C439. Small amounts of dUDP (dADP) are also observed during the interaction of UDP (ADP) with C225SR1R2 and are attributed to the presence of contaminating heterodimer of wt-Rl and mutant. A single mutation of an active site cysteine to a serine has converted the normal substrates into mechanism-based inhibitors. A mechanism to accommodate these amazing results is presented.

AB - Ribonucleotide reductase (RDPR) from Escherichia coli is composed of two subunits, Rl and R2, both of which are required to catalyze the conversion of nucleotides to deoxynucleotides. This reduction process is accompanied by oxidation of two cysteines within the active site to a disulfide. One of these putative active site cysteines, C225, has been mutated to a serine, and the properties of this mutant (C225SR1) have been investigated in detail. Incubation of C225SR1 and R2 with [3ʹ-3H, U-14C]UDP results in time-dependent inactivation of the enzyme! This inactivation is accompanied by production of 2.4 uracils, 3H2O, and 3H,14C-labeled protein with an absorbance change at 320 nm. There is an isotope effect (kH/K3H) on uracil production of 3.2. In addition, the tyrosyl radical on R2 is reduced. The observation of 3H20, indicative of 3ʹcarbon-hydrogen bond cleavage and loss of the tyrosyl radical, provides a direct test of our mechanistic hypothesis that cleavage of this bond occurs concomitantly with tyrosyl radical reduction. Incubation of [3ʹ-2H]UDP with C225SR1 and R2 resulted in a V and V/K isotope effect on loss of the radical of 2.0 and 2.0, respectively. These studies provide the first direct evidence for protein radical involvement in catalysis. Reduction of the tyrosyl radical on R2 is accompanied by a stoichiometric cleavage of the Rl polypeptide into two new polypeptides of 26 and 61 kDa. The 26-kDa polypeptide is the N-terminus of Rl, and hence cleavage of the polypeptide is occurring in the region of the mutation. The N-terminus of the 61-kDa polypeptide is blocked. The mutant RDPR is inactivated by three different mechanisms: loss of the tyrosyl radical on R2, alkylation of Rl by 2-methylene-3(2H)-furanone, and cleavage of Rl into two polypeptides. This sequence of events is also observed with the purine substrate ADP and requires the presence of the appropriate allosteric effector. Studies with a variety of single and double Rl mutants suggest that the cleavage reaction requires the presence of C462 and C439. Small amounts of dUDP (dADP) are also observed during the interaction of UDP (ADP) with C225SR1R2 and are attributed to the presence of contaminating heterodimer of wt-Rl and mutant. A single mutation of an active site cysteine to a serine has converted the normal substrates into mechanism-based inhibitors. A mechanism to accommodate these amazing results is presented.

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