Knut Feldmann

New devices for flow dialysis and ultrafiltration for the study of protein-ligand interactions

Abstract A flow dialysis procedure with an approach to steady state in less than half a minute has been developed. The main advantage of the new device is that it permits the use of lower levels of radioactivity and therefore allows determination of dissociation constants ( K D ) of tight complexes, i.e., with K D values of 10 −7 m or even lower. An ultrafiltration device was also developed which does not disturb binding equilibria because it uses only a small fraction of the ultrafiltrate (about 1% of the total solution) and which allows simultaneous measurements of six samples in a few minutes. Both methods were tested with rabbit skeletal muscle glycogen phosphorylases b and a (EC 2.4.1.1) and the allosteric ligand 5′-AMP. The K D values are in good agreement with those previously obtained by conventional methods.

Is the active form of pyridoxal-P in α-glucan phosphorylases a 5′-phosphate dianion?

The essential role of pyridoxal-5’-phosphate for the activity of all cY-glucan phosphorylases received strong support from recent studies with bacterial phosphorylases [ 11. The advantage of utilizing these enzymes for the study of the role of pyridoxal-P is obvious since they neither depend on allosteric effectors or covalent modification for the expression of activity [l-6]. The 5’-phosphate group of pyridoxalP is the sole phosphate moiety in catalytically active bacterial phosphorylases. Accordingly, ‘rP NMR spectra of Escherichia coli maltodextrin phosphorylase exhibit an exceptionally simple pattern. When compared with the 31P NMR spectra of rabbit skeletal muscle phosphorylase a and b [7] the resonances common to phosphorylase allow us to define the ionization state of the cofactor in the catalytically active enzyme.

Pyridoxal-5′-deoxymethylenephosphonate reconstituted D-serine dehydratase: A phosphorus-31 nuclear magnetic resonance study

Summary The phosphonate group of pyridoxal 5′-deoxymethylenephosphonate has a more favorable pK value than the phosphate group of pyridoxal-P to test the capabilities of this cofactor analogue to undergo specific ionic interactions with amino groups of model systems and amino acid side chains of D-serine dehydratase. Therefore, pyridoxal 5′-deoxymethylenephosphonate reconstituted, active D-serine dehydratase has been investigated using 31 P nuclear magnetic resonance (NMR) at 72.86 MHz. The 31 P chemical shift of the phosphonate group of the cofactor analogue is pH dependent with a pK a = 7.4, indicating exposure of the phosphonate group to solvent. Binding of the competitive inhibitor isoserine results in the formation of the isoserine-cofactor analogue complex. This transaldimination complex is fixed to the enzyme via a salt bridge of the dianionic phosphonate group of the cofactor analogue indicated by an apparent pK shift of the phosphonate group towards more acidic pH values. Similar shifts of the apparent pK value were observed for the corresponding Schiff base with n-dodecylamine when placed into a micellular environment. Addition of hexadecyltrimethylammonium bromide generating mixed micelles leads to intermediary pK values.

THE ROLE OF PYRIDOXALPHOSPHATE IN GLYCOGEN PHOSPHORYLASES

ABSTRACT 18 O exchange between the glucosyl and phosphoryl bridge oxygens of glucose-1-P with potato starch phosphorylase in the presence of cyclodextrins suggests that glycogen phosphorylases catalyze a double displacement reaction with retention of configuration involving a glucosyl-enzyme-intermediate (Ref. 1). In the framework of this catalytic mechanism and based on recent information on the three-dimensional structure of muscle glycogen phosphorylase (Ref. 2, 3), a possible role of the phosphate group of pyridoxal-5′-P either as proton- or as glucosyl-donor-acceptor group is discussed: