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2.4.2.1: purine-nucleoside phosphorylase

This is an abbreviated version!
For detailed information about purine-nucleoside phosphorylase, go to the full flat file.

Word Map on EC 2.4.2.1

Reaction

purine 2'-deoxyribonucleoside
+
phosphate
=
purine
 +
2-deoxy-alpha-D-ribose 1-phosphate

Synonyms

5'-deoxy-5'-methylthioadenosine phosphorylase II, 5'-methythioadenosine phosphorylase, adenine nucleoside phosphorylase, adenosine phosphorylase, AgPNP, AmPNP, ANP, ApMTAP, apPNP, bovPNP, bPNO, CKP1, cytokinin riboside phosphorylase, DeoD, deoD_1, DgPNP, EC592_06680, EcPNP, flB, GsPNPI, GsPNPII, GtPNP, hPNP, HpPNP, HsPNO, HsPNP, Ino-Guo phosphorylase, inosine phosphorylase, inosine-guanosine phosphorylase, KlacPNP, KLLA0_C16621g, MTAPII, MtbPNP, nucleotide phosphatase (2.4.2.1), PfPNP, phosphorylase, purine nucleoside, PiPNP, PNP, PNP 1, PNP 2, PNP I, PNP II, PNP-II, PNP-Zg, PNPase, pNPI, PNPII, Pu-NPase, punA, PUNP, PUNPI, PUNPII, purine deoxynucleoside phosphorylase, purine deoxyribonucleoside phosphorylase, purine nucleoside orthophosphate ribosyl transferase, purine nucleoside phosphorylase, purine nucleoside phosphorylase 1, purine nucleoside phosphorylase 2, purine nucleoside phosphorylase DeoD, purine nucleoside phosphorylase DeoD-type, purine nucleoside phosphorylase II, purine ribonucleoside phosphorylase, purine-nucleoside phosphorylases II, SalT, SSO2343, TgPNP, TTC0194, TTC1070, TtPNPI, TtPNPII, TvPNP, xanthosine phosphorylase, XAP, xapA, ylmD

ECTree

     2 Transferases
         2.4 Glycosyltransferases
             2.4.2 Pentosyltransferases
                2.4.2.1 purine-nucleoside phosphorylase

Engineering

Engineering on EC 2.4.2.1 - purine-nucleoside phosphorylase

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PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
F159W
the mutant shows reduced activity and stability compared to the wild type enzyme
F200W
the mutant shows reduced activity and stability compared to the wild type enzyme
L22E/H104R
-
mutation of human toward bovine purine nucleoside phosphorylase. Mutant is similar to human purine nucleoside phosphorylase in steady-state kinetic properties. Inhibitor DADMe-immucillin-G is an excellent mimic of the transition states for both human purine nucleoside phosphorylase and the mutant with a preference for the mutant. Thermodynamic parameters establish the mutant to be catalytically more efficient than the parent enzyme and reveal differences in the entropic component of catalysis
N123L/R210Q
mutation of bovine enzyme toward human enzyme. Steady-state kinetic studies indicate unchanged catalytic activity, the mutant enzyme has higher affinity for inhibitors that are mimics of a late dissociative transition state
N243D
V39T/N123L/R210Q
mutation of bovine enzyme toward human enzyme. Steady-state kinetic studies indicate unchanged catalytic activity, while presteady-state studies indicate that the chemical step is slower in the triple mutant. The mutant enzyme has higher affinity for inhibitors that are mimics of a late dissociative transition state. Mutant displays a highly dissociative SN1 transition state with low bond order to the leaving group, a transition state different from the native enzyme, with significant nucleophilic participation at the transition state
D204A
0.1% of wild-type activity towards inosine, adenosine and guanosine
D204A/R217A
about 0.2% of wild-type activity towards inosine, adenosine and guanosine
D204N
F159A
site-directed mutagenesis, the PNPF159A-FA complexes show a weak association of formycin A to the mutant's active center
F159Y
site-directed mutagenesis, a prominent quenching of the PNPF159Y emission indicates a complex formation, with the strongest association in the phosphate buffer, pH 7.0, relative to the wild-type enzyme. On the other hand, results testify to a deterioration of the interactions in the wild-type PNP/PNPF159Y mutant and formycin A complexes in the presence of the phosphate, pH 8.3
N239D
mutant enzyme shows no activity with the wild-type substrates inosine, xanthosine and guanosine. Unlike the wild-type enzyme, the mutant enzyme shows activity with adenosine
R117E/K121E/D139R
the mutant shows greater stability compared with the wild type dimer and changes in its structures compared with the wild type dimer in a hexamer
R117E/K121E/D139R/F120D
the mutant shows greater stability compared with the wild type dimer and changes in its structures compared with the wild type dimer in a hexamer
R117E/K121E/D139R/F120D/I128S/F131G
the mutant shows greater stability compared with the wild type dimer and changes in its structures compared with the wild type dimer in a hexamer
R117E/K121E/D139R/Y173S
the mutant shows greater stability compared with the wild type dimer and changes in its structures compared with the wild type dimer in a hexamer
R217A
about 0.5% of wild-type activity towards inosine and guanosine, 10.8% of activity towards adenosine
Y191L
mutant enzyme shows no activity with the wild-type substrate xanthosine. The ratio of Vmax to Km for inosine as substrate is 1.7fold lower than the ratio for wild-type enzyme
A117T
-
mutation leading to purine-nucleoside phosphorylase deficiency, occuring in two sisters
E201Q/N243D
F159G
mutant displays a strong increase in KM and modest decrease in kcat
F159Y
site-directed mutagenesis, pre-steadystate chemistry is reduced 32fold in mutant F159Y PNP. Pre-steady-state chemistry compares heavy and light molecular weight wild-type and mutant F159Y PNPs and finds a normal heavy-enzyme isotope effect of 1.31 for wild-type PNP and an inverse effect of 0.75 for F159Y mutant PNP. Increased isotopic mass in F159Y PNP causes more efficient transition state formation. Most heavy enzymes demonstrate normal heavy-enzyme isotope effects, and F159Y PNP is a rare example of an inverse effect
F200G
mutant displays a strong increase in KM and modest decrease in kcat
G51S
-
the mutant possesses wild type phosphorylase activity levels toward inosine and ribavirin
H257D
kcat/Km for inosine is 1460fold lower than wild-type value, greatly decreased affinity for Immucillin-H
H257F
kcat/Km for inosine is 68fold lower than wild-type value, greatly decreased affinity for Immucillin-H
H257G
kcat/Km for inosine is 610fold lower than wild-type value, greatly decreased affinity for Immucillin-H
H86A
-
10-25fold reduction in catalytic activity
K22E/H104R
L22E/H104R
-
mutation of human toward bovine purine nucleoside phosphorylase. Mutant is similar to human purine nucleoside phosphorylase in steady-state kinetic properties. Inhibitor DADMe-immucillin-G is an excellent mimic of the transition states for both human purine nucleoside phosphorylase and the mutant with a preference for the mutant. Thermodynamic parameters establish the mutant to be catalytically more efficient than the parent enzyme and reveal differences in the entropic component of catalysis
N243A
-
100fold decrease in turnover-number compared to wild-type enzyme
N243D
Q89A
-
10-25fold reduction in catalytic activity
Y88A
mutation introduced to render enzyme susceptible to substrate 2',3'-dideoxyinosine
Y88C
mutation introduced to render enzyme susceptible to substrate 2',3'-dideoxyinosine
Y88D
mutation introduced to render enzyme susceptible to substrate 2',3'-dideoxyinosine
Y88E
mutation introduced to render enzyme susceptible to substrate 2',3'-dideoxyinosine
Y88F
engineering of human enzyme to accept 2',3'-dideoxyinosine as substrate. Mutant displays 9-fold improvement in kcat and greater than 2-fold reduction in KM of 2',3'-dideoxyinosine
Y88H
mutation introduced to render enzyme susceptible to substrate 2',3'-dideoxyinosine
Y88I
mutation introduced to render enzyme susceptible to substrate 2',3'-dideoxyinosine
Y88K
mutation introduced to render enzyme susceptible to substrate 2',3'-dideoxyinosine
Y88L
mutation introduced to render enzyme susceptible to substrate 2',3'-dideoxyinosine
Y88M
mutation introduced to render enzyme susceptible to substrate 2',3'-dideoxyinosine
Y88N
mutation introduced to render enzyme susceptible to substrate 2',3'-dideoxyinosine
Y88Q
mutation introduced to render enzyme susceptible to substrate 2',3'-dideoxyinosine
Y88R
mutation introduced to render enzyme susceptible to substrate 2',3'-dideoxyinosine
Y88S
mutation introduced to render enzyme susceptible to substrate 2',3'-dideoxyinosine
Y88T
mutation introduced to render enzyme susceptible to substrate 2',3'-dideoxyinosine
Y88V
mutation introduced to render enzyme susceptible to substrate 2',3'-dideoxyinosine
Y88W
mutation introduced to render enzyme susceptible to substrate 2',3'-dideoxyinosine
N256D
site-directed mutagenesis, mutant KlacPNPN256D accepts both 6-oxopurines and 6-aminopurines as substrates, it shows the highest activity with adenosine
N256E
site-directed mutagenesis, the mutant is only active with inosine, no activity with guanosine, xanthosine, and adenosine
N256D
-
site-directed mutagenesis, mutant KlacPNPN256D accepts both 6-oxopurines and 6-aminopurines as substrates, it shows the highest activity with adenosine
-
N256E
-
site-directed mutagenesis, the mutant is only active with inosine, no activity with guanosine, xanthosine, and adenosine
-
N256D
-
site-directed mutagenesis, mutant KlacPNPN256D accepts both 6-oxopurines and 6-aminopurines as substrates, it shows the highest activity with adenosine
-
N256E
-
site-directed mutagenesis, the mutant is only active with inosine, no activity with guanosine, xanthosine, and adenosine
-
N256D
-
site-directed mutagenesis, mutant KlacPNPN256D accepts both 6-oxopurines and 6-aminopurines as substrates, it shows the highest activity with adenosine
-
N256E
-
site-directed mutagenesis, the mutant is only active with inosine, no activity with guanosine, xanthosine, and adenosine
-
N256D
-
site-directed mutagenesis, mutant KlacPNPN256D accepts both 6-oxopurines and 6-aminopurines as substrates, it shows the highest activity with adenosine
-
N256E
-
site-directed mutagenesis, the mutant is only active with inosine, no activity with guanosine, xanthosine, and adenosine
-
N256D
-
site-directed mutagenesis, mutant KlacPNPN256D accepts both 6-oxopurines and 6-aminopurines as substrates, it shows the highest activity with adenosine
-
N256E
-
site-directed mutagenesis, the mutant is only active with inosine, no activity with guanosine, xanthosine, and adenosine
-
N256D
-
site-directed mutagenesis, mutant KlacPNPN256D accepts both 6-oxopurines and 6-aminopurines as substrates, it shows the highest activity with adenosine
-
N256E
-
site-directed mutagenesis, the mutant is only active with inosine, no activity with guanosine, xanthosine, and adenosine
-
K244Q
-
ration of turnover-numer/Km is 83% of that for wild-type enzyme, no activity with adenosine
N243D
-
substitution results in an 8fold increase in Km-value for inosine and a 100fold decrease in ratio of turnover-number/Km. Catalyzes phosphorolysis of adenosine with a Km-value of 0.045 mM and ratio of turnover-number/Km 8fold that with inosine, wild-type enzyme shows no activity with adenosine
N243D/K244Q
-
14fold increase in Km-value for inosine and 7fold decrease in the ratio of turnover-number/Km as compared to wild-type enzyme. Phosphorolysis of adenosine with a Km-value of 0.042 mM and a ratio of turnover-number/Km twice that of the single D243D substitution
N243T
-
mutant enzyme shows no activity with adenosine
V66I
the mutant shows wild type activity
V66I/V73I
the mutant shows increased activity compared to the wild type enzyme
V66I/Y160F
the mutant shows increased activity compared to the wild type enzyme
V73I
the mutant shows wild type activity
V73I/Y160F
the mutant shows wild type activity
Y160F
the mutant shows reduced activity compared to the wild type enzyme
Y160F/V66I/V73I
the mutant has a 83fold decrease in turnover number for 5'-methylthioinosine with 2fold increase in Km value compared to the wild type enzyme
T156A
about 5fold decrease in kcat/Km
T156S
increase in kcat/Km
T90A
dramatic decrase in kcat/Km
T90A/T156A
500fold reduction in catalytic activity when compared with wild-type
T90R
increase in kcat/Km
T90R/T156S
1000fold enhancements in kcat/Km for inosine phosphorolysis
T90S
about 5fold decrease in kcat/Km
V206I
kcat/Km similar to wild-type
T156A
-
about 5fold decrease in kcat/Km
-
T90A
-
dramatic decrase in kcat/Km
-
T90A/T156A
-
500fold reduction in catalytic activity when compared with wild-type
-
T90R/T156S
-
1000fold enhancements in kcat/Km for inosine phosphorolysis
-
T90S
-
about 5fold decrease in kcat/Km
-
C254S/C256S
A196E/D238N
-
the mutations in the trimeric enzyme form change substrate specificity from guanosine to adenosine
N204D
-
the mutation in the hexameric enzyme form changes substrate specificity from adenosine to guanosine
N243D
-
site-directed mutagenesis, the mutation in trimeric PNP changes the substrate specificity, making 6-aminopurine nucleosides good substrates
A196E/D238N
Thermus thermophilus HB8 / ATCC 27634 / DSM 579
-
the mutations in the trimeric enzyme form change substrate specificity from guanosine to adenosine
-
N204D
Thermus thermophilus HB8 / ATCC 27634 / DSM 579
-
the mutation in the hexameric enzyme form changes substrate specificity from adenosine to guanosine
-
additional information