title: Relationship between enzyme specificity and the backbone dynamics of free and inhibited alpha-lytic protease.
abstract: To better understand the structural basis for the observed patterns in substrate specificity, the backbone dynamics of alpha-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 tauc was found to be 8.1 ns. Values of the spectral density function J(omega) at omega = 0, omegaN, and approximately omegaH 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 changes. A comparison of spectral densities for both the free and inhibited enzymes revealed that inhibitor binding preferentially stabilizes regions undergoing chemical exchange (which predominate around the active site) and only minimally affect regions of rapid motion. Slow motions, suggestive of backbone plasticity, are observed in most of the binding pocket residues. This may point to a mechanism for the observed broad specificity of the enzyme. The significance of the observed dynamics for substrate binding and specificity is discussed.
authors.affiliation: Graduate Group in Biophysics, Howard Hughes Medical Institute, University of California, San Francisco 94143-0448, USA.
authors.forename: J H
authors.initials: JH
authors.lastname: Davis
authors.forename: D A
authors.initials: DA
authors.lastname: Agard
mesh_terms: D001419:Bacteria / Q000201:enzymology; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D001897:Boronic Acids / Q000494:pharmacology; D018360:Crystallography, X-Ray; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D009842:Oligopeptides / Q000494:pharmacology; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000187:drug effects; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology*; D013379:Substrate Specificity / Q000187:drug effects; D013816:Thermodynamics
chemical_list: D001426:Bacterial Proteins; D001897:Boronic Acids; C109958:N-tert-butyloxycarbonyl-Ala-Pro-boroVal; D009842:Oligopeptides; D015842:Serine Proteinase Inhibitors; D012697:Serine Endopeptidases; C021826:myxobacter alpha-lytic proteinase