Cloning, expression, purification, cofactor requirements, and steady state kinetics of phosphoketolase-2 from Lactobacillus plantarum.
The genes xpk1 and xpk2(Delta1-21) encoding phosphoketolase-1 and (Delta1-7)-truncated phosphoketolase-2 have been cloned from Lactobacillus plantarum and expressed in Escherichia coli. Both gene-products display phosphoketolase activity on fructose-6-phosphate in extracts. A N-terminal His-tag construct of xpk2(Delta1-21) was also expressed in E. coli and produced active His-tagged (Delta1-7)-truncated phosphoketolase-2 (hereafter phosphoketolase-2). Phosphoketolase-2 is activated by thiamine pyrophosphate (TPP) and the divalent metal ions Mg(2+), Mn(2+), or Ca(2+). Kinetic analysis and data from the literature indicate the activators are MgTPP, MnTPP, or CaTPP, and these species activate by an ordered equilibrium binding pathway, with Me(2+)TPP binding first and then fructose-6-phosphate. Phosphoketolase-2 accepts either fructose-6-phosphate or xylulose-5-phosphate as substrates, together with inorganic phosphate, to produce acetyl phosphate and either erythrose-4-phosphate or glyceraldehyde-3-phosphate, respectively. Steady state kinetic analysis of acetyl phosphate formation with either substrate indicates a ping pong kinetic mechanism. Product inhibition patterns with erythrose-4-phosphate indicate that an intermediate in the ping pong mechanism is formed irreversibly. Background mechanistic information indicates that this intermediate is 2-acetyl-TPP. The irreversibility of 2-acetyl-TPP formation might explain the overall irreversibility of the reaction of phosphoketolase-2.
Yevenes
Alejandro
A
Department of Biochemistry, University of Wisconsin-Madison, 1710 University Avenue, Madison, WI 53726, USA.
Frey
Perry A
PA
D000446:Aldehyde-Lyases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D015870:Gene Expression; D007700:Kinetics; D048191:Lactiplantibacillus plantarum / Q000201:enzymology*; D010755:Organophosphates; D013403:Sugar Phosphates
D010755:Organophosphates; D013403:Sugar Phosphates; C026959:erythrose 4-phosphate; C011632:acetyl phosphate; D000446:Aldehyde-Lyases; C022367:phosphoketolase