Remote Mutations and Active Site Dynamics Correlate with Catalytic Properties of Purine Nucleoside Phosphorylase

¹ Albert Einstein College of Medicine

^* To whom correspondence should be addressed. E-mail: sschwartz{at}aecom.yu.edu.

Submitted on September 17, 2007
Revised on November 9, 2007
Accepted on 31 December 2007

	Abstract

It is found that upon mutating two surface residues in human purine nucleoside phosphorylase (PNP) (Lys22Glu and His104Arg) there is an enhancement of catalytic activity in the chemical step. This is true even though the mutations are quite remote from the active site, and there are no significant changes in crystallographic structure between the wildtype and mutant active sites. We propose that dynamic coupling from the remote residues to the catalytic site may play a role in catalysis, and it is this alteration in dynamics that causes an increase in chemical step rate. Computational results indicate that the mutant exhibits stronger coupling of promoting vibrations to the reaction coordinate than found in native human PNP. Power spectra comparing native and mutant proteins show a correlation between the vibrations of ImmG:O5'···ImmG:N4' and H257:N···ImmG:O5' consistent with a coupling of these motions. These modes are linked to the protein promoting vibrations. Stronger coupling of motions to the reaction coordinate increases the probability of reaching the transition state, and thus lowers the activation free energy. This motion has been shown to contribute to catalysis [Nunez, S.; Antoniou, D.; Schramm, V. L.; Schwartz, S. D. J. Am. Chem. Soc. 2004, 126, 15720-15729.] Coincident with the approach to the transition state, the sum of the distances of ImmG:O4'···ImmG:O5'···H257:N become smaller, stabilizing the oxacarbenium ion formed at the transition state. Combined results from crystallography, mutational analysis, chemical kinetics, and computational analysis are consistent with dynamic compression playing a significant role in forming the transition state. Stronger coupling of these pairs is observed in the catalytically enhanced mutant enzyme. That motion and catalysis are enhanced by mutations remote from the catalytic site, implicates dynamic coupling through the protein architecture as a component of catalysis in PNP.

Key Words: active site dynamics, catalysis, promoting vibration, remote mutation

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