Structural Characterization of the Reaction Pathway in Phosphoserine Phosphatase: Crystallographic “snapshots” of Intermediate States

• Published in: Journal of molecular biology 2002-05, Vol.319 (2), p.421-431
• Main Authors: Wang, Weiru, Cho, Ho S., Kim, Rosalind, Jancarik, Jaru, Yokota, Hisao, Nguyen, Henry H., Grigoriev, Igor V., Wemmer, David E., Kim, Sung-Hou
• Format: Article
• Language: English
• Subjects: Aluminum Compounds - metabolism; associative mechanism; Beryllium - metabolism; Binding Sites; Catalysis; Crystallography, X-Ray; dissociative mechanism; Fluorides - metabolism; Hydrogen-Ion Concentration; Magnesium - metabolism; Methanococcus - enzymology; Methanococcus jannaschii; Models, Molecular; phospho-aspartyl enzyme intermediate; Phosphoric Monoester Hydrolases - chemistry; Phosphoric Monoester Hydrolases - metabolism; phosphoserine phosphatase; phosphotransfer, phospho-monoester hydrolysis; Protein Structure, Secondary; Protein Structure, Tertiary; Temperature
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/S0022-2836(02)00324-8

Phosphoserine phosphatase (PSP) is a member of a large class of enzymes that catalyze phosphoester hydrolysis using a phosphoaspartate–enzyme intermediate. PSP is a likely regulator of the steady-state d-serine level in the brain, which is a critical co-agonist of the N-methyl- d-aspartate type of glutamate receptors. Here, we present high-resolution (1.5–1.9 Å) structures of PSP from Methanococcus jannaschii, which define the open state prior to substrate binding, the complex with phosphoserine substrate bound (with a D to N mutation in the active site), and the complex with AlF 3, a transition-state analog for the phospho-transfer steps in the reaction. These structures, together with those described for the BeF 3 − complex (mimicking the phospho-enzyme) and the enzyme with phosphate product in the active site, provide a detailed structural picture of the full reaction cycle. The structure of the apo state indicates partial unfolding of the enzyme to allow substrate binding, with refolding in the presence of substrate to provide specificity. Interdomain and active-site conformational changes are identified. The structure with the transition state analog bound indicates a “tight” intermediate. A striking structure homology, with significant sequence conservation, among PSP, P-type ATPases and response regulators suggests that the knowledge of the PSP reaction mechanism from the structures determined will provide insights into the reaction mechanisms of the other enzymes in this family.