Probing the catalytic allosteric mechanism of rabbit muscle pyruvate kinase by tryptophan fluorescence quenching.
Pyruvate kinase acts as an allosteric enzyme, playing a crucial role in the catalysis of the final step of the glycolytic pathway. In this study, site-specific mutagenesis and tryptophan fluorescence quenching were used to probe the catalytic allosteric mechanism of rabbit muscle pyruvate kinase. Movement of the B domain was found to be essential for the catalytic reaction. Rotation of the B domain in the opening of the cleft between domains B and A induced by the binding of activating cations allows substrates to bind, whereas substrate binding shifts the rotation of the B domain in the closure of the cleft. Trp-157 accounts for the differences in tryptophan fluorescence signal with and without activating cations and substrates. Trp-481 and Trp-514 are brought into an aqueous environment after phenylalanine binding.
Department of Chemistry, Fudan University, Shanghai, China.
Feng
F
Li
Ting
T
Yu
Yuwei
Y
Zhao
Shaoning
S
Yu
D000494:Allosteric Regulation; D000495:Allosteric Site; D000595:Amino Acid Sequence; D000818:Animals; D020134:Catalytic Domain; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009132:Muscles; D016297:Mutagenesis, Site-Directed; D011770:Pyruvate Kinase / Q000737:chemistry* / Q000235:genetics; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry*
D014364:Tryptophan; D011770:Pyruvate Kinase