Structural Basis by Which the N‑Terminal Polypeptide
Segment of Rhizopus chinensis Lipase Regulates Its
Substrate Binding Affinity
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Posted on 2019-09-12 - 14:33
Members
of an important group of industrial enzymes, Rhizopus lipases, exhibit valuable hydrolytic features that underlie their
biological functions. Particularly important is their N-terminal polypeptide
segment (NTPS), which is required for secretion and proper folding
but is removed in the process of enzyme maturation. A second common
feature of this class of lipases is the α-helical “lid”,
which regulates the accessibility of the substrate to the enzyme active
site. Some Rhizopus lipases also exhibit “interfacial
activation” by micelle and/or aggregate surfaces. While it
has long been recognized that the NTPS is critical for function, its
dynamic features have frustrated efforts to characterize its structure
by X-ray crystallography. Here, we combine nuclear magnetic resonance
spectroscopy and X-ray crystallography to determine the structure
and dynamics of Rhizopus chinensis lipase (RCL) with
its 27-residue NTPS prosequence (r27RCL). Both r27RCL and the truncated
mature form of RCL (mRCL) exhibit biphasic interfacial activation
kinetics with p-nitrophenyl butyrate (pNPB). r27RCL exhibits a substrate binding affinity significantly
lower than that of mRCL due to stabilization of the closed lid conformation
by the NTPS. In contrast to previous predictions, the NTPS does not
enhance lipase activity by increasing surface hydrophobicity but rather
inhibits activity by forming conserved interactions with both the
closed lid and the core protein structure. Single-site mutations and
kinetic studies were used to confirm that the NTPS serves as internal
competitive inhibitor and to develop a model of the associated process
of interfacial activation. These structure–function studies
provide the basis for engineering RCL lipases with enhanced catalytic
activities.
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Zhang, Meng; Yu, Xiao-Wei; Xu, Yan; Guo, Rey-Ting; Swapna, G. V. T.; Szyperski, Thomas; et al. (2019). Structural Basis by Which the N‑Terminal Polypeptide
Segment of Rhizopus chinensis Lipase Regulates Its
Substrate Binding Affinity. ACS Publications. Collection. https://doi.org/10.1021/acs.biochem.9b00462