Date of Graduation

8-2025

Document Type

Thesis

Degree Name

Master of Science in Chemistry (MS)

Degree Level

Graduate

Department

Chemistry & Biochemistry

Advisor/Mentor

Sakon, Joshua

Committee Member

Fan, Chenguang

Second Committee Member

Lay Jr. , Jackson

Third Committee Member

Nakanishi, Nagayasu

Keywords

Collagenase H; POU-IV Transcription Factor; Small Angle X-ray Scattering; Structural studies; X-ray crystallography

Abstract

Collagenase H (ColH) from Hathewaya histolytica plays a central role in collagen degradation through endopeptidase and carboxytripeptidase reactions. To better understand how its domain architecture supports these functions, we examined the solution structure of a catalytically inactive mutant, ColH-E416Q, using small-angle X-ray scattering (SAXS). The E416Q mutant is catalytically inactive because the glutamate residue at position 416, which normally functions as a general acid/base during catalysis, is replaced by glutamine—an amide that cannot donate or accept protons effectively, thus disabling the proton transfer steps essential for peptide bond hydrolysis. SAXS results indicate that the E416Q substitution leaves overall domain architecture and conformational behavior intact with comparative differences in dynamics. The E416Q mutation in ColH selectively abolishes catalytic activity while preserving substrate binding, making it a valuable tool for structural and mechanistic studies. By stabilizing enzyme–substrate complexes that would otherwise be transient, ColH-E416Q offers a strategic platform for capturing intermediate snapshots of collagen unwinding and cleavage. This presents a potential avenue for the development of novel collagenase inhibitors that target non-catalytic sites; an approach of particular interest given the close resemblance between bacterial catalytic sites and those of human matrix metalloproteases (MMPs), a similarity that made earlier inhibitor designs unsuitable due to anticipated side effects. These findings deepen our mechanistic understanding of bacterial collagenolysis and provide a structural basis for developing next-generation inhibitors or engineered collagenase variants with improved specificity.

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