Enhanced Preprints

Structural characterization of ligand binding and pH-specific enzymatic activity of mouse Acidic Mammalian Chitinase

  1. Roberto Efraín Díaz
  2. Andrew K. Ecker
  3. Galen J. Correy
  4. Pooja Asthana
  5. Iris D. Young
  6. Bryan Faust
  7. Michael C. Thompson
  8. Ian B. Seiple
  9. Steven J. Van Dyken
  10. Richard M. Locksley
  11. James S. Fraser author has email address
  1. Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, San Francisco, CA 94158, USA
  2. Tetrad Graduate Program, University of California, San Francisco, San Francisco, CA 94158, USA
  3. Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, CA 94158, USA
  4. Cardiovascular Research Institute, University of California, San Francisco, San Francisco, CA 94158, USA
  5. Chemistry and Chemical Biology Graduate Program, University of California, San Francisco, San Francisco, CA 94158, USA
  6. Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, CA 94158, USA
  7. Biophysics Graduate Program, University of California, San Francisco, San Francisco, CA 94158, USA
  8. Department of Chemistry and Chemical Biology, University of California, Merced, Merced, CA 95343, USA
  9. Department of Pathology and Immunology, Washington University School of Medicine in St.Louis, St. Louis, MO 63110, USA
  10. Department of Medicine, University of California, San Francisco, San Francisco, CA 94158, USA
  11. Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA 94143, USA
  12. University of California, San Francisco, Howard Hughes Medical Institute, San Francisco, CA 94143, USA

Peer review process

Not revised: This Reviewed Preprint includes the authors’ original preprint (without revision), an eLife assessment, and public reviews.

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Editors

  • Reviewing Editor
    Amie Boal
    Pennsylvania State University, University Park, United States of America
  • Senior Editor
    Qiang Cui
    Boston University, Boston, United States of America

Reviewer #1 (Public Review):

General comments:
This paper investigates the pH-specific enzymatic activity of mouse acidic mammalian chitinase (AMCase) and aims to elucidate its function's underlying mechanisms. The authors employ a comprehensive approach, including hydrolysis assays, X-ray crystallography, theoretical calculations of pKa values, and molecular dynamics simulations to observe the behavior of mouse AMCase and explore the structural features influencing its pH-dependent activity.

The study's key findings include determining kinetic parameters (Kcat and Km) under a broad range of pH conditions, spanning from strong acid to neutral. The results reveal pH-dependent changes in enzymatic activity, suggesting that mouse AMCase employs different mechanisms for protonation of the catalytic glutamic acid residue and the neighboring two aspartic acids at the catalytic motif under distinct pH conditions.

The novelty of this research lies in the observation of structural rearrangements and the identification of pH-dependent mechanisms in mouse AMCase, offering a unique perspective on its enzymatic activity compared to other enzymes. By investigating the distinct protonation mechanisms and their relationship to pH, the authors reveal the adaptive nature of mouse AMCase, highlighting its ability to adjust its catalytic behavior in response to varying pH conditions. These insights contribute to our understanding of the pH-specific enzymatic activity of mouse AMCase and provide valuable information about its adaptation to different physiological conditions.

Overall, the study enhances our understanding of the pH-dependent activity and catalytic properties of mouse AMCase and sheds light on its adaptation to different physiological pH environments.

Reviewer #2 (Public Review):

Summary: In this study of the mouse homolog of acidic mammalian chitinase, the overall goal is to provide a mechanistic explanation for the unusual observation of two pH optima for the enzyme. The study includes biochemical assays to establish kinetic parameters at different solution pH, structural studies of enzyme/substrate complexes, and theoretical analysis of amino acid side chain pKas and molecular dynamics.

Strengths: The biochemical assays are rigorous and nicely complemented by the structural and computational analysis. The mechanistic proposal that results from the study is well rationalized by the observations in the study.

Weaknesses: The overall significance of the work could be made more clear. Additional details could be provided about the limitations of prior biochemical studies of mAMC that warranted the kinetic analysis. The mouse enzyme seems unique in terms of its behavior at high and low pH, so it remains unclear how the work will enhance broader understanding of this enzyme class. It was also not clear can the findings be used for therapeutic purposes, as detailed in the abstract, if the human enzyme works differently.

  1. Howard Hughes Medical Institute
  2. Wellcome Trust
  3. Max-Planck-Gesellschaft
  4. Knut and Alice Wallenberg Foundation