UDP‐galactose 4‐epimerase from Kluyveromyces fragilis: existence of subunit independent functional site

Abstract

UDP-galactose 4-epimerase from Kluyveromyces fragilis is a stable homodimer of 75 kDa/subunit with noncovalently bound NAD acting as cofactor. Partial proteolysis with trypsin in the presence of 5 0 -UMP, a strong competitive inhibitor, led to a degraded product which was purified. Results from SDS-PAGE, size-exclusion (SE)-HPLC and ultracentrifugation indicated its monomeric status and size between 43 and 45 kDa. 'Two-step assay' with UDP-glucose dehydrogenase as coupling enzyme in the presence of NAD ensured epimerase activity of the monomer. The possibility of transient dimerization of monomeric epimerase during catalysis was excluded by SE-HPLC in the presence of excess substrate and NAD. This truncated enzyme retained catalytic site related properties like K m for UDP-galactose, 'NADH-like coenzyme fluorescence' and 'reductive inhibition' similar to its dimeric counterpart. Reversible reactivation of the monomer was achieved up to 95% within 3 min from 8 M urea induced unfolded state, indicating that the catalytic site could form independent of its quaternary structure. Equilibrium unfolding between 0 and 8 M urea indicated that the monomer was less stable compared to the dimer. Chemical modification of amino acids and reconstitution with etheno-NAD suggested that the architecture around the catalytic site of the monomer was conserved. Specific modification reagents further confirmed that the cysteine residues required for catalysis and coenzyme fluorophore reside exclusively on a single subunit negating a 'subunit sharing model' of its catalytic site.

Functional analysis of disease‐causing mutations in human UDP‐galactose 4‐epimerase

“…2). The kinetic parameters determined from these data (Table 1) are similar to those published for human GALE and for GALEs from other species [21,27,29,30]. There is no evidence that human GALE is glycosylated in vivo, and there is no anomalous migration of recombinant human GALE produced in yeast [27], and thus the observed activity probably reflects that of the native enzyme.…”

“…Crosslinking was carried out with N-(3dimethylaminopropyl)-N¢-ethylcarbodiimide (EDC), as described in the Experimental procedures. C, control lane containing 25 lM GALE not exposed to crosslinker; X, 25 lM GALE exposed to crosslinker; U, 25 lM GALE exposed to crosslinker in the presence of a saturating amount (1 mM) of UDP-galactose; M, molecular mass markers (29,45, 66, 97, 116 and 205 kDa). All the mutant proteins were exposed to crosslinker.…”

“…Then, 0.02 units of UDP‐glucose dehydrogenase (manufacturer's unit definition) were added and the absorbance at 340 nm was monitored for 2–3 min. This is necessary as commercial UDP‐galactose contains a small amount of UDP‐glucose [29]. Reactions were initiated by the addition of GALE (to a final concentration of 2.6 n m for the wild‐type protein and ranging from 2 n m to 330 n m for the mutants).…”

“…Proteolysis was carried out as described in the Experimental procedures, and then the products were separated by 15% SDS ⁄ PAGE and stained with Coomassie blue. C, control lanes containing 50 lM GALE; P, GALE digested with 90 nM thermolysin for 30 min at 37°C; U, GALE digested with 90 nM thermolysin for 30 min at 37°C in the presence of 1 mM UDP-galactose; M, molecular mass markers(29, 45, 66, 97, 116 and 205 kDa).…”

UDP‐galactose 4‐epimerase from Kluyveromyces fragilis – catalytic sites of the homodimeric enzyme are functional and regulated

“…They can function independently as an epimerase and a mutarotase [11]. Interestingly, when trypsin digestion is performed in the presence of only the epimerase inhibitor, the mutarotase domain is fragmented, yielding a 45 kDa monomeric epimerase [11,13].…”