On the Conformation of Pyridoxal Phosphate Imine in Solution and in Aspartate‐Aminotransferase Active Site

Abstract

The work comprises : 1. Determination by nuclear magnetic resonance method of the conformation of the imino group of pyridoxal-P oxime in solution. It has been found to be nearly coplanar with the aromatic ring.2. Theoretical calculation by the method of atom-atom potentials of the permitted conformations of pyridoxal-P imine and its analogs : 6-methylpyridoxal-P and 5'-methylpyridoxal-P. There have been found seven favourable conformations for pyridoxal-P imine, some of which were prohibited in the analogs mentioned.3. Experimental estimation of the conformation of pyridoxal-Pimine in the active site of aspartate aminotransferase with the use of the above sterically hindered analogs and 5'-deoxy-5'-carboxymethylenepyridoxal analog. In result, the number of the possible conformations for pyridoxal-P imine in the active site has been reduced to a pair of symmetrical conformations.Pyridoxal5'-phosphate, the coenzyme of aspartate aminotransferase, is present in the active site of the enzyme in the form of an aldimine linked to the epsilon amino group of a lysine residue [l].The conformation of coenzyme moiety of the aldimine is specified by the following rotation angles around ordinary bonds: C, -C,, (x), C, -C,, (cp), 0 -C,, ($), and P -0 (0) (Fig. 1). An angle of 0" corresponds to a cis position of the bonds, adjoining the rotation axis. An increase in the magnitude of the angle corresponds to a clockwise rotation of a distant bond if one looks along the rotation axis. The cis positions are depicted in Fig. 1 .X-Ray analysis data of the crystals of pyridoxal-P and pyridoxamine-P derivatives [2 -61 have been recently published. The oxime of pyridoxal-P was shown to have a flat, competely extended conformation (1 = o", cp = 357" (-3"), $ = 174") [6]. In the derivatives lacking a double bond in position 4, the phosphate group may be present in one of several favourable conformations depending on the nature of the substituent in position 4 [2-61. The conformation of pyridoxal-P imines in solution was shown to depend on the ionization state of the polar groups of the molecule [7].Since pyridoxal-P is constrained by the apoenzyme by all its side groups [8], only one conformation of the molecule is present on the protein. Knowledge of this particular conformation is necessary prerequisite for understanding the real mechanism of enzymatic transamination.

Conformational analysis of pyridoxal Schiff's bases. Nuclear magnetic resonance studies of the conformations about the C4-C4', Cα-Cβ and N-Cα bonds of the pyridoxal Schiff's bases of amino acids

“…hand, previous data (Heinert & Martell, 1962) have indicated the cis conformation, in agreement with recent nuclear Overhauser effect (NOE) measurements and theoretical calculations for pyridoxal phosphate and its oxime (A, W = CH*OP03*-, X = N, Y = CH3, Z = OH) (Tumanyan et al, 1974). An x-ray structure analysis of pyridoxal phosphate oxime also shows the cis conformation (Barrett & Palmer, 1969).…”

The Molecular Evolution of Pyridoxal‐5′‐Phosphate‐Dependent Enzymes

“…The great majority of amino acid substitutions at the active site of B, enzymes have two mutually opposite kinetic consequences: a decrease in the rate of the specific reaction and an increase in the rates of side reactions. Clearly, the reaction specificity of PLP-dependent enzymes and possibly of many other enzymes is not only achieved by accelerating the specific reaction but also by preventing potential side reactions (Retey, 1990;Vacca et al, 1997). Enhancement of the catalytic efficiency of engineered enzymes and catalytic antibodies requires subtle adaptations in the active-site topochemistry as they Modern reaction and substrate-specific 86 enzymes Figure 13.…”

Stereochemical constraint in the evolution of pyridoxal‐5′‐phosphate‐DApenDAnt enzymes. A hypothesis

“…All rights reserved. SSDI SO0 14-5793(96)00298-0 P. Christen et al IFEBS Letters 389 (1996) 12 [8,9].) assisted deprotonation at Ca, an integral step of transamination and many other reactions catalyzed by B6 enzymes [7].…”