Open AccessApplication of the Induced‐Transport Test to the Mechanism of Pig‐Kidney Phosphoglyceromutase
Author(s) -
Britton Hubert G.,
Carrears José,
Grisolia Santiago
Publication year - 1973
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1973.tb02935.x
Subject(s) - chemistry , isomerization , substrate (aquarium) , stereochemistry , enzyme , cotransporter , intramolecular force , phosphate , biophysics , biochemistry , catalysis , organic chemistry , sodium , oceanography , biology , geology
The induced transport test has been used to study the mechanism of pig‐kidney phosphoglyceromutase (2,3‐bisphospho‐ d ‐glycerate: 2‐phospho‐ d ‐glycerate phosphotransferase). At intermediate substrate concentration (0.5 mM) there was cotransport of 32 P‐labelled substrates and no induced transport of 14 C‐labelled substrates. These findings, excluded an intramolecular transfer of phosphate or an exchange of phosphate between two or more molecules of substrate, but were quantitatively in accord with a phosphoenzyme (ping pong) mechanism with a rapid isomerisation of the “phosphoenzyme”; or with an intermolecular transfer of phosphate from 2,3‐bisphosphoglycerate to the substrates (the sequential mechanism) if there were a slow isomerisation of the enzyme · bisphosphoglycerate complex. Similar results at low substrate concentrations (9.5 μM) however excluded the latter possibility since it would have required a K m for 3‐phosphoglycerate of less than 1.9 μM compared with a measured value of about 270 μM. The enzyme therefore has a phosphoenzyme mechanism. A lack of induced transport with 14 C‐labelled substrates at high substrate concentrations indicated a rate constant for isomerisation of the “phosphoenzyme” in excess of ∼ 10 6 s −1 and the absence of induced transport with 14 C‐labelled substrates over a wide concentration range excluded certain types of interaction between active centres. To explain the extremely rapid isomerisation of the phosphoenzyme a modified mechanism is proposed in which there is no formal isomerisation of the phosphoenzyme. The nature of the “phosphoenzyme” is discussed and it is suggested that it is probably a true phosphoenzyme with the phosphate covalently bound to the protein. The possible evolutionary relationship of this mechanism to that of a 2,3‐bisphosphoglycerate phosphatase is discussed.