The mechanism of addition of pyridoxal 5'-phosphate to Escherichia coli apo-serine hydroxymethyltransferase.
Previous studies suggest that the addition of pyridoxal 5'-phosphate to apo-serine hydroxymethyltransferase from Escherichia coli is the last event in the enzyme's folding process. We propose a mechanism for this reaction based on quenched-flow, stopped-flow and rapid-scanning stopped-flow experiments. All experiments were performed with an excess of apo-enzyme over cofactor, since excess pyridoxal 5'-phosphate results in a second molecule of cofactor binding to Lys346, which is part of the tetrahydropteroylglutamate-binding site. The equilibrium between the aldehyde and hydrate forms of the cofactor affects the kinetics of addition to the active site. Direct evidence of the formation of an intermediate aldimine between the cofactor and the active-site lysine was obtained. The results have been interpreted according to a three-step mechanism in which: (i) both aldehyde and hydrate forms of the cofactor bind rapidly and non-covalently to the apo-enzyme; (ii) only the aldehyde form reacts with the active-site lysine to give an intermediate internal aldimine with unusual spectral properties; and (iii) a final conformational change gives the native holo-enzyme.
Malerba
Francesca
F
Dipartimento di Scienze Biochimiche A. Rossi Fanelli, Università degli Studi di Roma La Sapienza, Piazzale Aldo Moro 5, 00185 Rome, Italy.
Bellelli
Andrea
A
Giorgi
Alessandra
A
Bossa
Francesco
F
Contestabile
Roberto
R
D001051:Apoenzymes / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D012696:Glycine Hydroxymethyltransferase / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D017510:Protein Folding; D011732:Pyridoxal Phosphate / Q000378:metabolism*; D014803:Vitamin B Complex / Q000378:metabolism*
D001051:Apoenzymes; D029968:Escherichia coli Proteins; D014803:Vitamin B Complex; D011732:Pyridoxal Phosphate; D012696:Glycine Hydroxymethyltransferase