Mass spectrometric and kinetic studies on slow progression of papain-catalyzed polymerization of L-glutamic acid diethyl ester.
Papain polymerizes L-glutamic acid diethyl ester (Glu-di-OEt) regioselectively, resulting in the formation of poly (gamma-ethyl alpha-L-glutamic acid) with various degrees of polymerization of less than 13. Reaction temperatures below 20 degrees C were appropriate for the reaction in terms of suppression of non-enzymatic degradation of Glu-di-OEt and an increase in the peptide yield, while the reaction was preceded by a pronounced induction period. Mass spectrometric analyses of the reaction conducted at 0 degrees C revealed that the accumulation of the initial dimerization product, L-glutamyl-L-glutamic acid triethyl ester (Glu-Glu-tri-OEt), was limited during the induction period, and that a sequential polymer derived from a further elongation of the dimer was the tetramer, but not the trimer. Kinetic analyses of acyl transfer reactions with Glu-di-OEt and Glu-Glu-tri-OEt as acyl acceptors and Nalpha-benzoyl-L-arginine ethyl ester as an acyl donor affirmed that Glu-Glu-tri-OEt bound more strongly than Glu-di-OEt both to the S- and S'-subsites of papain. Therefore, what occurred during the initial stage of the polymerization was interpreted as follows: the rate of the papain-catalyzed dimerization of Glu-di-OEt was extremely slow, once Glu-Glu-tri-OEt was initially synthesized it exclusively bound to the active site of papain, and then papain utilized the dimer in polymerization effectively rather than the monomer.
Narai-Kanayama
Asako
A
Faculty of Applied Life Science, Nippon Veterinary and Life Science University, Musashino-shi, Tokyo 180-8602, Japan. a-narai@nvlu.ac.jp
Koshino
Hiroyuki
H
Aso
Keiichi
K
D002384:Catalysis; D005971:Glutamates / Q000737:chemistry*; D006358:Hot Temperature; D007700:Kinetics; D013058:Mass Spectrometry; D008956:Models, Chemical; D010206:Papain / Q000737:chemistry*; D011091:Polyesters / Q000138:chemical synthesis* / Q000737:chemistry
D005971:Glutamates; D011091:Polyesters; C011829:glutamic acid diethyl ester; D010206:Papain