Spin Labeling Studies of d‐Glyceraldehyde‐3‐phosphate Dehydrogenase

Abstract

~-Glyceraldehyde-3-phosphate dehydrogenase has been reacted with spin labels, which were designed to elucidate the geometry of the active site of the enzyme. Three series of spin labels were used, which represented either substrate and coenzyme analogs or irreversible inhibitors, the separation of the active site directing part from the nitroxide radical being readily adjusted through the length of the intervening methylene chain.The effect of this variation on the electron paramagnetic resonance spectrum of the labeled protein as well as the effect of coenzyme binding on these spectra have been investigated. All spectra are complex and represent the superposition of a t least three different types of spectra. These three types of spectra are correlated to nitroxide radicals in three different types of motion: (a) very fast rotation in a fluid region, (b) medium fast rotation of a hindered radical and (c) slow rotation of an almost immobilized radical. The spin labels differ only in the relative population in the three states related to the three types of motion. Also addition of the coenzyme NAD only alters this relative population. A spin labeled coenzyme analog, which has the nitroxide in the region of the sugar-nicotinamide bond, is bound to the protein and displays an electron paramagnetic resonance spectrum typical for an immobilized radical. The geometry of the active of ~-glyceraldehyde-3-phosphate dehydrogenase is complex and cannot be described as a simple cleft, but instead as a system of narrow and wider spaces, which is changed by the binding of the coenzyme.The spin labeling technique has been introduced by McConnell [l]. It has been applied to a wide spectrum of problems and has been especially successful in biochemical and biological applications. Reviews on spin labels have appeared [Z, 31. Spin labels have been used in biochemistry to detect conformational changes of haemoglobin [3,4], aspartate transcarbamylase [5] as well as to study the activity [6] and the geometry of the active site [7] of ol-chymotrypsin. Spin labels were used in this work in an attempt to determine conformational and geometrical properties of the tetrameric enzyme D-glyceraldehyde-3-phosphate dehydrogenase. ~-Glyceraldehyde

Interaction of purified human transcortin with spin labelled steroids

“…(ii) Being most sensitive to the local environment of the spin label, the ESR parameters can characterize a binding site (2T value). Thus, two simultaneous binding systems may be distinguished (as in the case of HSA \ and HCBG); this approach might be developped into a powerful tool by a mathematical treatment of the superimposed mixed spectra [25].…”