Cadstatins (CSs), (gamma-Glu-Cys)n-Ala polymers derived from cysthione (gamma-Glu-Cys-Ala) by the action of the enzyme CS syntha
se, play a pivotal role in heavy metal tolerance in some animals by chelating toxic metal ions, such as Cd2+, and decreasing their free concentrations. CSS-catalyzed CS synthesis from cysthione proceeds by the transpeptidation of a gamma-Glu-Cys (gammaEC) unit from one cysthione molecule to another to form CS2 (= (gammaEC)2A) and, after the accumulation of sufficient (substrate) levels of CSs, by the transpeptidation of a gammaEC unit from cysthione or a CS to another CS (CSn) molecule to form CSn+1. Appropriately, CS synthase has been defined as a gamma-Glu-Cys dipeptidyl transpeptidase and thought to catalyze a reaction of the type: (gammaEC)A + (gammaEC)nA ⇌ (gammaEC)(gammaEC)nA + A Implicit, therefore, has been the assumption that CS chain extension proceeds C to N with cleavage of the Cys-Ala peptide bond of the donor, not the acceptor.
A. Attempt to draw an outline of the two steps of the ping-pong mechanism of gamma-Glu-Cys dipeptidyl transpeptidase, assuming it is similar to a serine protease.
B. Suppose that you have recently cloned a cDNA encoding the enzyme CSS and have a ready supply of pure catalytically active recombinant enzyme. The enzyme comprises a single polypeptide species of Mr 22,000 which, alone, is sufficient for catalysis. Assume that you have ample supplies of [3H-Ala]cysthione (cysthione with the 3H label on the Ala residue, alone), and [35S-Cys] cysthione (cysthione with the 35S label on the Cys residue alone) and unlabeled cysthione. When high total concentrations of CSS ([CSS]t’s) are added to media containing [3H-Ala]cysthione and the appearance of free [3H]Ala in the incubation medium is measured, the results in the figure shown below are obtained. Note the non-zero intercepts on the ordinate, which you suspect represent extremely rapid [3H]Ala release (initial release at a rate exceeding the time-resolution of your measurements). How do you account for the kinetics of [3H]Ala release? Propose other experiments to further test your proposal.
C. A property of CSS that you initially find confusing is that substitution of any one of its 41 Ser residues to Ala or Cys by site-directed mutagenesis has little or no effect on catalytic activity. Disappointed by the negative outcome of these experiments, you decide to mutate each of the 15 Cys residues to Ser or Ala residues instead. In so doing you find that substitution of Cys91 to Ser only partially abolishes rapid [3H]Ala release whereas an Ala substitution at the same position completely abolishes it. Substitution of any one of the other 14 Cys residues with Ser or Ala has little or no effect on activity. On the basis of these results and the time-dependence of free Ala release shown below, propose a model for the role(s) played by Cys91 in catalysis that is capable of explaining the partial activity of C91S mutants and complete lack of activity of C91A mutants. Note that while C91S-mutated enzyme elicits a seemingly instantaneous release of free [3H]Ala from [3H-Ala]cysthione, C91A-mutated enzyme does not. Neither C91S nor C91A mutated enzyme mediate the slower steady rate of [3H]Ala release seen after the initial burst.
