Determination of the Full Catalytic Cycle among Multiple
Cyclophilin Family Members and Limitations on the Application of CPMG-RD
in Reversible Catalytic Systems
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Abstract
Cyclophilins catalyze <i>cis</i> ↔ <i>trans</i> isomerization of peptidyl–prolyl
bonds, influencing protein
folding along with a breadth of other biological functions such as
signal transduction. Here, we have determined the microscopic rate
constants defining the full enzymatic cycle for three human cyclophilins
and a more distantly related thermophilic bacterial cyclophilin when
catalyzing interconversion of a biologically representative peptide
substrate. The cyclophilins studied here exhibit variability in on-enzyme
interconversion as well as an up to 2-fold range in rates of substrate
binding and release. However, among the human cyclophilins, the microscopic
rate constants appear to have been tuned to maintain remarkably similar
isomerization rates without a concurrent conservation of apparent
binding affinities. While the structures and active site compositions
of the human cyclophilins studied here are highly conserved, we find
that the enzymes exhibit significant variability in microsecond to
millisecond time scale mobility, suggesting a role for the inherent
conformational fluctuations that exist within the cyclophilin family
as being functionally relevant in regulating substrate interactions.
We have additionally modeled the relaxation dispersion profile given
by the commonly employed Carr–Purcell–Meiboom–Gill
relaxation dispersion (CPMG-RD) experiment when applied to a reversible
enzymatic system such as cyclophilin isomerization and identified
a significant limitation in the applicability of this approach for
monitoring on-enzyme turnover. Specifically, we show both computationally
and experimentally that the CPMG-RD experiment is sensitive to noncatalyzed
substrate binding and release in reversible systems even at saturating
substrate concentrations unless the on-enzyme interconversion rate
is much faster than the substrate release rate