Multi-step Mechanism in the Hydrolysis of Pteroylpolyglutamates by Chicken Intestine

Abstract

Abstract Upon incubation with a crude extract of chicken intestine, the disappearance of pteroylglutamyl-γ-hexaglutamate (PteGlu7) was found to be accompanied first by the formation of pteroylglutanyl-γ-glutamate (PteGlu2) and γ-Glu5. On prolonged incubation PteGlu, γ-Glu4, γ-Glu3, and Glu also appeared. These findings indicate that the catalytic hydrolysis of PteGlu7 involves a multi-step reaction. Three distinct enzymes participating in the catalysis were separated and fully or partially purified from the crude chicken intestine extract. Each of these enzymes catalyzes one distinct step of the over-all reaction. One of the enzymes, which has been purified, catalyzes the hydrolysis of PteGlu7 to PteGlu2 and γ-Glu5. A second enzyme, which was found to be a sulfhydryl enzyme, catalyzes the hydrolysis of PteGlu2 to PteGlu and Glu. The third enzyme of interest catalyzes the hydrolysis of γ-Glu5 and other γoligoglutamyl peptides. PteGlu2 and γ-l-Glu5, the intermediary products, were synthesized and used as initial substrates for the three enzymes described above. These substrates were hydrolyzed independently by the relevant enzymes. Kinetic analysis of the formation of products of hydrolysis of PteGlu7 in the presence of crude extract of chicken intestine showed that PteGlu2 and PteGlu are formed at different rates. The rate of the formation of PteGlu2, the first product, was linear with respect to time until a stationary stage was reached; after prolonged incubation the concentration of PteGlu2 diminished. PteGlu appeared only after a lag time which was absent when PteGlu2 was used as starting substrate. The time required for the appearance of this stationary stage in respect to PteGlu2 and for the lag time in appearance of PteGlu were found to be inversely proportional to the concentration of the enzyme protein. The results quoted here fulfill the requirements for the following consecutive reaction pathway: PteGlu7(E1)/→ PteGlu2 + Glu5 (1) PteGlu2(E2)/→ PteGlu + Glu (2) Glu5(E3)/→ Glu4 + Glu (3) Glu4(E3)/→ Glu3 + Glu (4)