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Relationship between Enzyme Specificity and the Backbone Dynamics of Free and Inhibited R-Lytic Protease,

Summary: Relationship between Enzyme Specificity and the Backbone Dynamics of Free and
Inhibited R-Lytic Protease,
Jonathan H. Davis§ and David A. Agard*
Graduate Group in Biophysics, Howard Hughes Medical Institute, and Department of Biochemistry and Biophysics,
UniVersity of California, San Francisco, 513 Parnassus AVenue, San Francisco, California 94143-0448
ReceiVed December 2, 1997; ReVised Manuscript ReceiVed March 26, 1998
ABSTRACT: To better understand the structural basis for the observed patterns in substrate specificity, the
backbone dynamics of R-lytic protease have been investigated using 15N relaxation measurements. The
enzyme was inhibited with the peptide boronic acid N-tert-butyloxycarbonyl-Ala-Pro-boroVal [Kettner,
C. A., et al. (1988) Biochemistry 27, 7682], which mimics interactions occurring in the tetrahedral transition
state or nearby intermediates, and the dynamics of the unbound and inhibited enzyme were compared.
Arrayed 2-D NMR spectra were acquired to measure T1, T2, and steady-state {1H}-15N NOE of >95%
of the backbone amides in both protein samples. The overall rotational correlation time c was found to
be 8.1 ns. Values of the spectral density function J() at ) 0, N, and H were derived from the
relaxation results using reduced spectral density mapping [Ishima, R., & Nagayama, K. (1995) Biochemistry
34, 3162]. The resultant spectral densities were interpreted to indicate regions of fast motion (nanosecond
to picosecond) and of intermediate chemical exchange (millisecond to microsecond). The protein has 13
regions with increased motion on the fast time scale; these generally fall on exterior turns and loops and
most correlate with regions of higher crystallographic B-factors. Several stretches of backbone undergo
intermediate chemical exchange, indicating motion or other processes that cause temporal chemical shift


Source: Agard, David - Department of Biochemistry and Biophysics, University of California at San Francisco


Collections: Biotechnology; Biology and Medicine