Characterization of histidinol phosphate aminotransferase from Escherichia coli.
Histidinol phosphate aminotransferase (HPAT) is a pyridoxal 5'-phosphate (PLP)-dependent aminotransferase classified into Subgroup I aminotransferase, in which aspartate aminotransferase (AspAT) is the prototype. In order to expand our knowledge on the reaction mechanism of Subgroup I aminotransferases, HPAT is an enzyme suitable for detailed mechanistic studies because of having low sequence identity with AspAT and a unique substrate recognition mode. Here we investigated the spectroscopic properties of HPAT and the effect of the C4-C4' strain of the PLP-Lys(214) Schiff base on regulating the Schiff base pK(a) in HPAT. Similar to AspAT, the PLP-form HPAT showed pH-dependent absorption spectral change with maxima at 340 nm at high pH and 420 nm at low pH, having a low pK(a) of 6.6. The pK(a) value of the methylamine-reconstituted K214A mutant enzyme was increased from 6.6 to 10.6. Mutation of Asn(157) to Ala increased the pK(a) to 9.2. Replacement of Arg(335) by Leu increased the pK(a) to 8.6. On the other hand, the pK(a) value of the N157A/R335L double mutant enzyme was 10.6. These data indicate that the strain of the Schiff base is the principal factor to decrease the pK(a) in HPAT and is crucial for the subsequent increase in the Schiff base pK(a) during catalysis, although the electrostatic effect of the arginine residue that binds the negatively charged group of the substrate is larger in HPAT than that in AspAT. Our findings also support the idea that the strain mechanism is common to Subgroup I aminotransferases.
Mizuguchi
Hiroyuki
H
Department of Biochemistry, Osaka Medical College, Takatsuki 569-8686, Japan.
Hayashi
Hideyuki
H
Miyahara
Ikuko
I
Hirotsu
Ken
K
Kagamiyama
Hiroyuki
H
D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D007700:Kinetics; D012545:Schiff Bases; D055672:Static Electricity; D000637:Transaminases / Q000737:chemistry* / Q000378:metabolism
D029968:Escherichia coli Proteins; D012545:Schiff Bases; D000637:Transaminases; C053430:histidinol-phosphate aminotransferase