Nathan J. Roth

QUATERNARY STRUCTURE, MG2+ INTERACTIONS, AND SOME KINETIC PROPERTIES OF THE BETA -GALACTOSIDASE FROM THERMOANAEROBACTERIUM THERMOSULFURIGENES EM1

Theβ-galactosidase fromThermoanaerobacterium thermosulfurigenes EM1 was found to be a dimer with a monomer molecular weight of about 85,000. It lacks theα-peptide and an importantα-helix that are both needed for dimer-dimer interaction and there is no homology in other important dimer-dimer interaction areas. These differences in structure probably account for the dimeric (rather than tetrameric) structure. Only 0.19 Mg2+ bound per monomer and Mg2+ had only small effects on the activity and heat stability. The absence of residues equivalent to Glu-416 and His-418 (two of the three ligands to Mg2+ in theβ-galactosidase fromEscherichia coli) probably accounts for the low level of Mg2+ binding and the consequent lack of response to Mg2+. Both Na+ and K+ also had no effect on the activity. The enzyme activity witho-nitrophenyl-β-D-galactopyanoside (ONPG) was very similar to that withp-nitrophenyl-β-D-β-D-galactopyranoside (PNPG) and the ONPG pH profile was very similar to the PNPG pH profile. These differences are in contrast to theE. coli β-galactosidase, which dramatically discriminates between these two substrates. The lack of discrimination by theT. thermosulfurigenes β-galactosidase could be due to the absence of the sequence equivalent to residues 910-1023 of theE. coli β-galactosidase. Trp-999 is probably of the most importance. Trp-999 of theE. coli β-galactosidase is important for aglycone binding and ONPG and PNPG differ only in their aglycones. The suggestion that the aglycone site of theT. thermosulfurigenes β-galactosidase is different was strengthened by competitive inhibition studies. Compared toE. coli β-galactosidase, D-galactonolactone was a very good inhibitor of theT. thermosulfurigenes enzyme, while L-ribose inhibited poorly. These are transition-state analogs and the results indicate thatT. thermosulfurigenes β-galactosidase binds the transition state differently than doesE. coli β-galactosidase. Methanol and glucose were good acceptors of galactose, and allolactose was formed when glucose was the acceptor. Allolactose could not, however, be detected by TLC when lactose was the substrate. The differences noted may be due to the thermophilic nature ofT. thermosulfurigenes.

β-galactosidases (Escherichia coli) with double substitutions show that Tyr-503 acts independently of Glu-461 but cooperatively with Glu-537

Abstractβ-Galactosidases with single substitutions for Tyr-503, Glu-461, and Glu-537 and with double substitutions for Tyr-503 and either Glu-461 or Glu-537 were constructed. Control experiments showed that the very low kcat values obtained for the double-substituted enzymes were not a result of contamination, reversion, or nonactive site activity catalyzed on the surface of the proteins. Circular dichroism studies showed that the structures of the enzymes were intact. E461Q/Y503F-β-galactosidase was inactivated in an “additive” manner. This indicated that Glu-461 and Tyr-503 act independently in catalysis. Because these residues are at opposite sides of the active site and act in different steps, this is expected. E537D/Y503F-β-galactosidase was only inactivated a few-fold more than the most inactive of its two single-substituted constituent β-galactosidases. This showed that Glu-537 and Tyr-503 interact cooperatively on the same step. This correlates well with the proposed role of Tyr-503 as an acid catalyst for the breakage of the covalent bond between Glu-537 and galactose.

A Rapid Method for the Purification of Large Amounts of an α-Complementing Peptide Derived from β-Galactosidase (E. coli)

Abstract A DNA segment coding for residues 6–44 of β-galactosidase was ligated to a vector with the glutathione-S-transferase gene which also contained a sequence coding for a thrombin recognition site. The fused protein, with an additional 9 amino acids coded for by the vector, was purified using a glutathione agarose affinity column. A peptide made up of residues 6–44 of β-galactosidase and the 9 additional amino acids was then cleaved from the glutathione-S-transferase using thrombin and purified with a gel filtration column. The peptide was about 3–4 times as active for α-complementation as the α-peptide derived from CNBr digestion of wild type β-galactosidase.

Probing the roles of conserved histidine residues in β-galactosidase (E. coli) using site directed mutagenesis and transition state analog inhibition

Publisher Summary Histidine (His) residue often play important roles in the structure and function of enzymes, and is often found within the active site as an acid/base catalyst or plays roles in modulating conformational changes. This chapter illustrates and determines the functional roles of His-357 and His-391 of β-galactosidase, without structural data. The results suggest that His-357 and His-391 are required for proper transition state stabilization and may form direct inter-actions with a planar galactosyl transition state intermediate, and that His-357 and His-391 might be the residues within the active site of 6-galactosidase, which mediate some of these interactions. As His-357 and His-391 are conserved in both the β-galactosidase and β-glucuronidase family, it is unlikely that either of these His is involved in interactions with the 4- hydroxyl position, because this hydroxyl is axial in galactose but is equatorial in glucuronic acid. The crystal structure of β-galactosidase shows that His-357 and His-391 are near the known active site residues and probably line the active site cavity. Therefore, they have the potential to form direct interactions with the substrate in the transition state form.