Interrelations between electronic structure and spatial geometry of specific ligands in the functioning active site of some pyridoxal‐p‐dependent enzymes

Abstract

Using a spectrokinetic approach the absorption spectra of the short-lived transient products in the enzymatic reaction of the glutamate decarboxylase with natural substrate are determined for the first time. The quantum-chemical calculations of the electronic structure and spectra of various ionic species of numerous vitamin Bb derivatives allowed a hypothesis on the nature of the intermediate products detected to be suggested. A model of the enzyme functioning taking into account the charge equilibria and some electronic-conformational relations is proposed.The impressive current advances in enzymology are dependent, to a considerable degree, on the active attention to this branch of science on the part of investigators working in a variety of other fields. In fact, solution of the most topical problems in biomedical sciences cannot, at present, be achieved without a thorough understanding of the mechanisms of enzyme action.A large and very important group of enzymes are those requiring pyridoxal-5'-phosphate as cofactor and effecting a variety of transformations of amino acids in all types of organisms-the so-called PLP-enzymes [l]. A large number of studies have been devoted to the investigation of enzymes belonging to this group, with studies aiming at elucidation of the mechanism of enzymic transamination reactions as a predominant topic.As opposed to our previous communication [ 2 ] , where a tentative model was formulated for the mechanism of enzymatic a-amino acid decarboxylation, the present paper discusses the interaction of glutamate decarboxylase from Escherichia coli with its natural substrate, taking into consideration the charge equilibria of coenzyme and substrate. The electronic structures of some nonenzymatic (model) coenzyme-substrate complexes are discussed. A more correct assignment to possible intermediates is suggested for the absorption bands recorded by means of the stop-flow procedure. (In the earlier paper [2] the spectroscopic resolution rested on the assumption that all spectral changes at 420 nm and beyond this wavelength were due only to the decrease in amount of the "internal" Schiff base; this resulted in somewhat inexact evaluation of the long-wave band in the difference spectrum.) In addition, some general aspects of the catalytic action of pyridoxal enzymes will be discussed.It should be stated that in the postulations regarding the model of enzymatic decarboxylation, the electronic structure and total energy, in the ground state, of

The Roles of His-167 and His-275 in the Reaction Catalyzed by Glutamate Decarboxylase from Escherichia coli

“…The slow conversion of the wild-type enzyme to an unreactive complex during the first few seconds of reaction, is not detectable in the H167N mutant. The absorbance changes that the cofactor undergoes in the first few seconds after mixing with glutamate have been reported earlier (21,4) and it has been shown that the chromophore formed absorbs maximally at 345 nm (4). However, the kinetic constants characterizing the different phases were not determined and it was not shown that the process leads to a less active form of the enzyme.…”

“…It was noticeable that, using the conditions described in Ref. 4 (pyridine HCl as buffer), the rate of the reactions catalyzed by the H167N mutant was only about 30% less than that of the wild-type enzyme compared with 50% in acetate buffer.…”

“…An additional protonation occurs in a side reaction where a proton is added to C4Ј of the cofactor rather than C␣ of the substrate to give an external aldimine of pyridoxamine phosphate with succinic semialdehyde. This reaction, which occurs only once in 3 ϫ 10 5 turnovers in E. coli glutamate decarboxylase (4), is a feature of most amino acid decarboxylases (5)(6)(7)(8)(9). In some of these enzymes this abortive transamination is kinetically more prominent and is almost certainly metabolically important because it inactivates the enzyme by leaving the cofactor as pyridoxamine phosphate.…”

The application of glutamic acid α-decarboxylase for the valorization of glutamic acid

“…The cause of the decrease of GAD activity with time is known to be related to a side reaction that takes place once every 300,000 turnovers, which is an abortive transamination reaction. 20,21 In this case, GAD replaces a carbonyl group of PLP with an amine group from a GABA molecule, thereby producing pyridoxamine-5-phosphate (PMP) and succinic semialdehyde. PMP dissociates from the active site, leaving GAD in its apoform, which is conformationally less stable.…”

Ab initio theory of hydrogen bonding in vitamin B6

“…I), namely pyridoxine (R = CH20H), pyridoxm i n e (R = CH2NH2), and pyridoxal (R = CHO). It has been suggested [2] that enzymes, based on this vitamin, may exhibit their biological activity in vivo when the vitamin is protonized. Various tautomeric forms have been postulated [3,4] for this vitamin.…”