Identification of basic residues involved in substrate binding and catalysis by pyrophosphate-dependent phosphofructokinase from Propionibacterium freudenreichii.

Despite overall low identity of sequence between ATP- and PPi-dependent phosphofructo-1-kinases (PFK), an alignment permits the tentative identification of catalytically important residues in PPi-dependent PFK. Seven basic residues of the PPi-dependent PFK of Propionibacterium freudenreichii, Arg-310, Lys-315, Arg-326, Arg-186, Arg-78, Arg-90, and Lys-148 were chosen on the basis of this alignment for site-directed mutagenesis to neutral residues. Mutants R186A, K315A, and R326A had substantial increases of 43-, 389- and 678-fold, respectively, in Km for fructose 6-phosphate relative to wild-type enzyme and relatively small effects on kcat. The modest kinetic effects of mutations at Arg-78, Arg-90, and Arg-310 were not considered to indicate important roles for these residues. On the other hand, mutant K148M had a Km for PPi that was increased by 132-fold and kcat lowered by a factor of 490. Cho and Cook (Cho, Y.-K., and Cook, P.F. (1988) Biochemistry 28, 4155-4160) concluded on the basis of kinetic and chemical modification data that the enzyme utilizes a proton shuttle mechanism and that two lysines are involved in substrate binding. The present data along with our previous data on mutant D151A support a double proton shuttle mechanism involving Asp-151 and Lys-148 with Lys-148, Arg-326, Lys-315, and perhaps Arg-186 being important for substrate binding.