Poster: Enzymology

Abs # 254: Kinetic analysis of plant-type 5’-adenylylsulfate reductase, a sulfate assimilation enzyme

Presenter:	Leustek, Thomas , leustek@aesop.rutgers.edu
Authors	Leustek, Thomas  (A)   Bick, Julie-Ann  (A)   Rahman, Afroza  (A)   Kim, Sung-Kun  (B)   Knaff, David  (B)
Affiliations:	(A): Rutgers University (B): Texas Rech University

5-adenylylsulfate (APS) reductase catalyzes the formation of sulfite from APS using reduced glutathione as electron donor. This enzyme is believed to be essential for cysteine biosynthesis in plants. Plant APS reductase has a bipartite primary structure consisting of a domain with homology to bacterial CysH, thioredoxin-dependent APS and 5-phosphoadenylylsulfate (PAPS) reductases, and a C-terminal domain with homology to the redox active proteins thioredoxin and glutaredoxin. The bacterial enzymes are believed to have a ping-pong catalytic mechanism. The catalytic mechanism of the enzyme from Arabidopsis thaliana was explored with initial velocity, end-product inhibition, and enzyme inactivating reagents. The Michaelis constants for reduced glutathione and APS are 0.83 mM and 0.38 mM respectively, and the enzyme has a Vmax of 1.25 mmoles min-1 mg protein-1. Initial velocity and product inhibition studies were consistent with an ordered Bi-Bi mechanism in which a ternary enzyme-substrate complex is formed. Reduced glutathione is the first reactant to bind and oxidized glutathione is the last product released. Iodoacetamide, a thiolate reactive agent, potently inactivates APS reductase only if the enzyme is reduced first with reduced glutathione of dithiothreitol. This result suggets that enzyme reduction exposes a thiolate group that is essential for catalytic activity.