Structural Characterization of pH-Dependent Enzymatic Activity and Ligand Binding of Mouse Acidic Mammalian Chitinase
Kernekoncepter
Mouse Acidic Mammalian Chitinase (mAMCase) exhibits dual pH optima at pH 2.0 and 6.5, allowing it to function in both acidic and neutral environments. Structural, biochemical, and computational analyses reveal how mAMCase employs distinct mechanisms to protonate its catalytic glutamate residue depending on the environmental pH.
Resumé
The content describes a comprehensive study of the structural and biochemical properties of mouse Acidic Mammalian Chitinase (mAMCase), a key enzyme involved in chitin degradation and innate immune response.
The key highlights are:
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Kinetic analysis revealed that mAMCase exhibits dual pH optima, with maximum activity at pH 2.0 and a secondary optimum at pH 6.5, unlike the human homolog which has a single optimum at pH 4.6.
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Structural studies of mAMCase in complex with chitin oligomers identified extensive conformational heterogeneity in ligand binding, including the discovery of non-canonical "n+0.5" sugar-binding subsites.
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Analysis of the catalytic triad (Asp136-Asp138-Glu140) showed that the orientation of the Asp138 residue is correlated with ligand occupancy in the active site. Theoretical pKa calculations suggested that Asp138 can adopt different protonation states to facilitate proton transfer to the catalytic Glu140 at acidic versus neutral pH.
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Molecular dynamics simulations supported the hypothesis that mAMCase employs distinct mechanisms for protonating Glu140 at low pH (direct protonation from solution) versus neutral pH (proton shuttling via Asp138).
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The dual pH optima of mAMCase are proposed to arise from its ability to access different protonation states of the catalytic triad, allowing it to function effectively in both the acidic stomach environment and the more neutral lung environment.
These findings provide a refined understanding of the catalytic mechanism governing mAMCase activity across different pH conditions, which may enable the engineering of improved enzyme variants for therapeutic applications.
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Structural characterization of ligand binding and pH-specific enzymatic activity of mouse Acidic Mammalian Chitinase
Statistik
The rate of 4MU-chitobioside catalysis by mAMCase catalytic domain is 6.3-fold higher at pH 2.0 compared to pH 7.4.
The Michaelis-Menten constant (KM) of mAMCase catalytic domain is 2.5-fold higher at pH 2.0 compared to pH 7.4.
The catalytic efficiency (kcat/KM) of mAMCase catalytic domain is highest at pH 6.5.
Citater
"mAMCase exhibits two distinct pH optima, which is unlike most enzymes that exhibit a shift or broadening of enzymatic activity across conditions."
"We hypothesize that these activity data resemble two overlapping activity distributions, suggesting that the rate at lower pH activity is dependent on the concentration of free protons in solution and that the higher pH optimum results from a distinct mechanism."
"Together these data support a model in which mAMCase employs two different mechanisms for obtaining a proton in a pH-dependent manner, providing a refined explanation as to how this enzyme recognizes its substrate in disparate environments."
Dybere Forespørgsler
How do the structural differences between mouse and human AMCase homologs contribute to their distinct pH activity profiles?
The structural differences between mouse and human AMCase homologs play a significant role in their distinct pH activity profiles. One key aspect is the presence of ionizable residues in mouse AMCase that are not present in human AMCase. These ionizable residues likely contribute to the overall stability of mouse AMCase and may influence its enzymatic activity at different pH levels. Mutations in human AMCase, such as Lys78Gln, Asp82Gly, and Lys160Gln, result in the loss of surface-stabilizing salt bridges, potentially affecting its activity at acidic pH. Additionally, previous studies have identified specific mutations in human AMCase that could impact the pKa of key residues in the catalytic triad, leading to differences in pH-dependent activity between the two homologs.
What are the potential implications of mAMCase's dual pH optima for its physiological roles in the stomach and lungs?
The dual pH optima of mAMCase have important implications for its physiological roles in the stomach and lungs. In the stomach, where the pH is around 2.0, mAMCase exhibits high enzymatic activity, allowing it to efficiently degrade chitin from ingested food. This acidic pH optimum ensures effective chitin clearance in the stomach environment. On the other hand, in the lungs where the pH is closer to 7.0, mAMCase still maintains significant enzymatic activity, indicating its ability to function in a more neutral environment. This dual pH activity profile suggests that mAMCase has evolved to perform its chitin-degrading function optimally in diverse chemical conditions, highlighting its importance in maintaining pulmonary health by clearing chitin and preventing accumulation in the airways.
Could the insights into mAMCase's pH-dependent catalytic mechanism be leveraged to engineer enzymes with tunable pH activity for therapeutic applications?
The insights gained from studying mAMCase's pH-dependent catalytic mechanism hold great potential for engineering enzymes with tunable pH activity for therapeutic applications. By understanding how mAMCase adapts its catalytic mechanism to different pH environments, researchers can potentially design and engineer enzymes that exhibit similar dual pH optima. This tunable pH activity could be beneficial for developing enzyme variants that are more effective in specific physiological conditions or disease contexts where pH plays a critical role. For example, by modulating the pH optima of enzymes involved in chitin degradation, it may be possible to enhance their therapeutic efficacy in conditions like asthma, lung fibrosis, or other chitin-related disorders. This approach could open up new opportunities for developing targeted enzyme therapies tailored to different pH microenvironments within the body.
