Study of Nucleophile Binding in the Penicillin Acylase Active Center. Kinetic Analysis

M. I. Youshko, A. L. Bukhanov, and V. K. Svedas^*

Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, 119992 Russia; fax: (095) 938-2422; E-mail: vytas@belozersky.msu.ru

^* To whom correspondence should be addressed.

Received February 28, 2002; Revision received March 20, 2002

---

The influence of the external nucleophile (6-aminopenicillanic acid) on the kinetics of the penicillin acylase-catalyzed acyl transfer reactions was studied using a highly sensitive spectrophotometric assay. An adequate kinetic scheme is suggested based on kinetic analysis of the experimental dependencies of the k_cat and K_m values on the nucleophile concentration. The proposed kinetic scheme has been verified by a quantitative description of the above-mentioned experimental dependencies using the set of kinetic parameters obtained from independent experiments. Such an approach can be used for modeling of different penicillin acylase-catalyzed acyl transfer reactions.

---

KEY WORDS: penicillin acylase, nucleophile binding, kinetic analysis

---