Altered thermodynamics from remote mutations altering human toward bovine purine nucleoside phosphorylase.
Biochemistry
; 47(8): 2559-64, 2008 Feb 26.
Article
em En
| MEDLINE
| ID: mdl-18281956
ABSTRACT
Human (HsPNP) and bovine (BtPNP) purine nucleoside phosphorylases are homotrimers with the catalytic sites located near the subunit-subunit interfaces. Despite the high amino acid sequence similarity (87% identical) and the fully conserved catalytic site contacts between BtPNP and HsPNP, crystal structures reveal that the subunits interact differently and isotope effect studies indicate distinct transition-state structures. The subunit interfaces and crystallographic packing properties of BtPNP differ from HsPNP. Hypothetically, mutating HsPNP toward BtPNP might alter the dynamic, catalytic and subunit packing properties of HsPNP to become more similar to BtPNP. Amino acids Lys22 and His104 in HsPNP were target candidates based on crystal packing contacts and were replaced with their BtPNP counterparts to give Lys22GluHis104Arg (ER-PNP). The kinetic properties (steady and pre-steady state), inhibition constants, and thermodynamic properties of ER-PNP were compared to HsPNP and BtPNP. The ER-PNP is similar to HsPNP in steady-state kinetic properties. However HsPNP and ER-PNP show remarkable ratios for (Km guanosine)/(Ki* DADMe-ImmG) of 2.8 x 10(70 and 4.7 x 10(7) respectively, suggesting that DADMe-ImmG is an excellent mimic of the transition states for both HsPNP and ER-PNP with a preference for ER-PNP. Thermodynamic parameters obtained from the temperature dependence studies of the chemical step establish ER-PNP to be catalytically more efficient than the parent enzyme and reveal differences in the entropic component of catalysis. The two companion manuscripts (Luo, M., Li, L. and Schramm, V. L. (2008) Biochemistry 47, 2565-2576; Li, L., Luo, M., Ghanem, M., Taylor, E. A., and Schramm, V. L. (2008) Biochemistry 47, 2577-2583) report changes in transition-state structure as a consequence of mutations remote from the catalytic sites of both HsPNP and BtPNP.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Purina-Núcleosídeo Fosforilase
/
Mutação
Limite:
Animals
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Humans
Idioma:
En
Ano de publicação:
2008
Tipo de documento:
Article