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Literature summary for 2.7.7.19 extracted from

  • Balbo, P.B.; Bohm, A.
    Mechanism of poly(A) polymerase: Structure of the enzyme-MgATP-RNA ternary complex and kinetic analysis (2007), Structure, 15, 1117-1131.
    View publication on PubMedView publication on EuropePMC

Crystallization (Commentary)

Crystallization (Comment) Organism
mutant D154A in complex with MgATP-RNA, hanging drop vapour diffusion method, in 0.1 M bis-Tris propane, pH 6.4, 0.2 M Li-acetate, and 16% PEG 3350 Saccharomyces cerevisiae
mutant D154A, trapped in complex with ATP and a five residue poly(A). Enzyme has undergone significant domain movement and shows a closed conformation with extensive interactions between substrates and all three polymerase domains Saccharomyces cerevisiae

Protein Variants

Protein Variants Comment Organism
D154A crystallization data, closed conformation with extensive interactions between substrates and all three polymerase domains Saccharomyces cerevisiae
D154A the mutant has a nearly identical melting temperature as wild type PAP Saccharomyces cerevisiae
K215A 2- to 4fold increase in Km values Saccharomyces cerevisiae
K215A the mutant has a nearly identical melting temperature as wild type PAP Saccharomyces cerevisiae
N189A residue bridges the N and middle domains in the closed state Saccharomyces cerevisiae
N189A the mutant has a nearly identical melting temperature as wild type PAP Saccharomyces cerevisiae
N226A mutation affects the equilibrium between the open- and closed-domain forms of the enzyme Saccharomyces cerevisiae
N226A the mutant has a nearly identical melting temperature as wild type PAP Saccharomyces cerevisiae
Y224F mutation affects the equilibrium between the open- and closed-domain forms of the enzyme Saccharomyces cerevisiae
Y224F altered melting temperature (44°C) compared to the wild type enzyme Saccharomyces cerevisiae

General Stability

General Stability Organism
substrates poly(A) and Mg-ATP induce the conformational change, resulting in stabilization of the closed enzyme state and enabling catalysis Saccharomyces cerevisiae

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.036
-
ATP wild-type, pH 7.0, 30°C Saccharomyces cerevisiae
0.0468
-
(A)n wild-type, pH 7.0, 30°C Saccharomyces cerevisiae
0.055
-
GTP mutant N226A, pH 7.0, 30°C Saccharomyces cerevisiae
0.062
-
GTP wild-type, pH 7.0, 30°C Saccharomyces cerevisiae
0.08
-
ATP mutant N189A, pH 7.0, 30°C Saccharomyces cerevisiae
0.104
-
CTP wild-type, pH 7.0, 30°C Saccharomyces cerevisiae
0.106
-
(A)n mutant N189A, pH 7.0, 30°C Saccharomyces cerevisiae
0.14
-
CTP mutant N226A, pH 7.0, 30°C Saccharomyces cerevisiae
0.148
-
CTP mutant N189A, pH 7.0, 30°C Saccharomyces cerevisiae
0.195
-
(A)n mutant K215A, pH 7.0, 30°C Saccharomyces cerevisiae
0.249
-
ATP mutant N226A, pH 7.0, 30°C Saccharomyces cerevisiae
0.367
-
(A)n mutant N226A, pH 7.0, 30°C Saccharomyces cerevisiae
0.368
-
CTP mutant K215A, pH 7.0, 30°C Saccharomyces cerevisiae
0.406
-
ATP mutant K215A, pH 7.0, 30°C Saccharomyces cerevisiae
0.711
-
(A)n mutant Y224F, pH 7.0, 30°C Saccharomyces cerevisiae
0.929
-
ATP mutant Y224F, pH 7.0, 30°C Saccharomyces cerevisiae
4.7
-
CTP mutant Y224F, pH 7.0, 30°C Saccharomyces cerevisiae

Metals/Ions

Metals/Ions Comment Organism Structure
Mg2+ essential for activity Saccharomyces cerevisiae

Organism

Organism UniProt Comment Textmining
Saccharomyces cerevisiae P29468
-
-

Purification (Commentary)

Purification (Comment) Organism
nickel affinity column chromatography and ion exchange chromatography Saccharomyces cerevisiae

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
(A)n + ATP
-
Saccharomyces cerevisiae (A)n+1 + diphosphate
-
?
(A)n + CTP
-
Saccharomyces cerevisiae (A)n-C + diphosphate
-
?
(A)n + diphosphate
-
Saccharomyces cerevisiae (A)n-1 + ATP
-
?
(A)n + GTP
-
Saccharomyces cerevisiae (A)n-G + diphosphate
-
?
ATP + RNA
-
Saccharomyces cerevisiae diphosphate + RNA(A)n
-
?

Synonyms

Synonyms Comment Organism
PAP PAP is a template-independent polymerase that belongs to the DNA polymerase beta family of enzymes Saccharomyces cerevisiae
poly(A) polymerase
-
Saccharomyces cerevisiae

Temperature Stability [°C]

Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
30 48 remains stable at 30°C, the melting temperature for the wild type enzyme is at 48°C Saccharomyces cerevisiae