EFFECT OF REDOX ENVIRONMENT ON THE IN-VITRO AND IN-VIVO FOLDING OF RTEM-1 BETA-LACTAMASE AND ESCHERICHIA COIL ALKALINE-PHOSPHATASE

The oxidative folding mechanisms of two Escherichia coil periplasmic p roteins, alkaline phosphatase and RTEM-1 beta-lactamase, have been exa mined in vitro and in vivo. In contrast to eukaryotic proteins, which require a relatively reducing environment for optimal folding rates, b oth alkaline phosphatase and beta-lactamase fold fastest under very ox idizing conditions. For example, bovine pancreatic ribonuclease exhibi ts an optimal folding rate in a redox buffer consisting of 1 mM GSH an d 0.2 mM GSSG (Lyles, M. M., and Gilbert, H. F (1991) Biochemistry 30, 613-619); however, both E. coil alkaline phosphatase and beta-lactama se exhibit optimal in vitro folding rates at low concentrations of GSH (< 0.4 mM) and very high concentrations of GSSG (4-8 mM). For both ba cterial proteins, GSH inhibits oxidative folding. Under optimal redox conditions, the rate-limiting step for the in vitro oxidative folding of alkaline phosphatase depends on the concentration of the protein, c onsistent with a mechanism involving rapid oxidation followed by slow dimerization. With beta-lactamase, the oxidative folding mechanism inv olves a competition between disulfide bond formation and folding of th e molecule into a catalytically active conformation that buries the 2 reduced cysteines in the core of the enzyme. The effects of including a thiol reductant in the growth medium on the in vivo folding of alkal ine phosphatase and beta-lactamase are similar to the effects observed during in vitro folding of these enzymes. The levels of both oxidized proteins are decreased by GSH in the growth medium. However, addition of a disulfide oxidant to the growth medium does not positively affec t the production of either enzyme. These observations are consistent w ith the idea that the oxidative folding mechanisms of E. coil periplas mic proteins and, by inference, proteins of the eukaryotic endoplasmic reticulum have evolved to accommodate constraints placed on the foldi ng reaction by the folding environment, The consequences of difference s between the folding mechanisms in eukaryotic and prokaryotic disulfi de containing proteins on the expression of eukaryotic proteins in the bacterial periplasm are discussed.