2ON6

Crystal structure of human purine nucleoside phosphorylase mutant H257F with Imm-H

Summary for 2ON6

• Entry DOI: 10.2210/pdb2on6/pdb
• Related: 2A0W 2A0X 2A0Y 2OC4 2OC9
• Descriptor: Purine nucleoside phosphorylase, 1,4-DIDEOXY-4-AZA-1-(S)-(9-DEAZAHYPOXANTHIN-9-YL)-D-RIBITOL (3 entities in total)
• Functional Keywords: purine nucleoside phosphorylase, transferase
• Biological source: Homo sapiens (human)
• Cellular location: Cytoplasm, cytoskeleton : P00491
• Total number of polymer chains: 1
• Total formula weight: 32460.16

Authors

Rinaldo-Matthis, A.,Murkin, A.S.,Almo, S.C.,Schramm, V.L. (deposition date: 2007-01-23, release date: 2007-05-22, Last modification date: 2024-05-22)

Primary citation

Murkin, A.S.,Birck, M.R.,Rinaldo-Matthis, A.,Shi, W.,Taylor, E.A.,Almo, S.C.,Schramm, V.L.
Neighboring Group Participation in the Transition State of Human Purine Nucleoside Phosphorylase
Biochemistry, 46:5038-5049, 2007

PubMed Abstract: The X-ray crystal structures of human purine nucleoside phosphorylase (PNP) with bound inosine or transition-state analogues show His257 within hydrogen bonding distance of the 5'-hydroxyl. The mutants His257Phe, His257Gly, and His257Asp exhibited greatly decreased affinity for Immucillin-H (ImmH), binding this mimic of an early transition state as much as 370-fold (Km/Ki) less tightly than native PNP. In contrast, these mutants bound DADMe-ImmH, a mimic of a late transition state, nearly as well as the native enzyme. These results indicate that His257 serves an important role in the early stages of transition-state formation. Whereas mutation of His257 resulted in little variation in the PNP x DADMe-ImmH x SO4 structures, His257Phe x ImmH x PO4 showed distortion at the 5'-hydroxyl, indicating the importance of H-bonding in positioning this group during progression to the transition state. Binding isotope effect (BIE) and kinetic isotope effect (KIE) studies of the remote 5'-(3)H for the arsenolysis of inosine with native PNP revealed a BIE of 1.5% and an unexpectedly large intrinsic KIE of 4.6%. This result is interpreted as a moderate electronic distortion toward the transition state in the Michaelis complex with continued development of a similar distortion at the transition state. The mutants His257Phe, His257Gly, and His257Asp altered the 5'-(3)H intrinsic KIE to -3, -14, and 7%, respectively, while the BIEs contributed 2, 2, and -2%, respectively. These surprising results establish that forces in the Michaelis complex, reported by the BIEs, can be reversed or enhanced at the transition state.

PubMed: 17407325
DOI: 10.1021/bi700147b

Experimental method

X-RAY DIFFRACTION (2.503 Å)