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beta-Galactosidase alpha-complementation. Overlapping sequences.

J K Welply, A V Fowler, I Zabin

    The Journal of Biological Chemistry
    |July 10, 1981
    PubMed
    Summary
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    Defective beta-galactosidase (M15 and M112) enzyme activity is restored by complementation with specific peptide fragments. This structural complementation restores tetrameric structure and enzyme function, highlighting key regions for beta-galactosidase activity.

    Area of Science:

    • Enzymology
    • Protein Biochemistry
    • Molecular Biology

    Background:

    • Beta-galactosidase is a crucial enzyme involved in lactose metabolism.
    • Defective forms, M15 and M112 proteins, lack specific amino acid residues and exhibit reduced or no enzymatic activity.
    • Understanding the structural basis of beta-galactosidase function is vital for enzyme engineering and therapeutic applications.

    Purpose of the Study:

    • To investigate the structural and functional complementation of defective beta-galactosidase variants (M15 and M112 proteins) using specific peptide fragments.
    • To identify the essential regions within beta-galactosidase responsible for restoring enzymatic activity and proper quaternary structure.
    • To elucidate the structural dynamics and accessibility of different regions in both defective and complemented beta-galactosidase forms.

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    Main Methods:

    • Complementation assays involving incubation of M15 and M112 proteins with specific beta-galactosidase peptide fragments (CNBr2, 3-41 peptide).
    • Structural analysis of complemented enzymes, including determination of quaternary structure (dimer to tetramer transition).
    • Limited proteolysis studies using trypsin to identify the N-terminal segment and assess conformational differences between defective and complemented enzymes.

    Main Results:

    • Enzyme activity was restored to M15 and M112 proteins upon incubation with beta-galactosidase fragments, forming functional tetrameric enzymes.
    • A smaller alpha-donor peptide (3-41) effectively complemented M15 protein, indicating M15 can provide residues 42-92.
    • Trypsin treatment revealed that the alpha-donor peptide forms the N-terminus of the complemented enzyme, and complemented enzymes exhibit a more closed, stable structure compared to the defective proteins.

    Conclusions:

    • Complementation with specific peptide fragments can restore both the enzymatic activity and the native tetrameric structure of defective beta-galactosidase.
    • The study precisely maps the contribution of different peptide segments to the overall structure and function of beta-galactosidase.
    • Defective beta-galactosidase variants display more open conformations, making them susceptible to proteolysis, while complemented enzymes are more stable.