Role of Lysine Versus Arginine in Enzyme Cold-adaptation: Modifying Lysine to Homo-arginine Stabilizes The Cold-adapted α-amylase from Pseudoalteramonas Haloplanktis

Document Type


Publication Date


Digital Object Identifier (DOI)



The cold-adapted α-amylase from Pseudoalteromonas haloplanktis (AHA) is a multidomain enzyme capable of reversible unfolding. Cold-adapted proteins, including AHA, have been predicted to be structurally flexible and conformationally unstable as a consequence of a high lysine-to-arginine ratio. In order to examine the role of low arginine content in structural flexibility of AHA, the amino groups of lysine were guanidinated to form homo-arginine (hR), and the structure–function–stability properties of the modified enzyme were analyzed by transverse urea gradient-gel electrophoresis. The extent of modification was monitored by MALDI-TOF-MS, and correlated to changes in activity and stability. Modifying lysine to hR produced a conformationally more stable and less active α-amylase. The kcat of the modified enzyme decreased with a concomitant increase in ΔH# and decrease in Km. To interpret the structural basis of the kinetic and thermodynamic properties, the hR residues were modeled in the AHA X-ray structure and compared to the X-ray structure of a thermostable homolog. The experimental properties of the modified AHA were consistent with K106hR forming an intra-Domain B salt bridge to stabilize the active site and decrease the cooperativity of unfolding. Homo-Arg modification also appeared to alter Ca2+ and Cl− binding in the active site. Our results indicate that replacing lysine with hR generates mesophilic-like characteristics in AHA, and provides support for the importance of lysine residues in promoting enzyme cold adaptation. These data were consistent with computational analyses that show that AHA possesses a compositional bias that favors decreased conformational stability and increased flexibility. Proteins 2006. © 2006 Wiley-Liss, Inc.

Was this content written or created while at USF?


Citation / Publisher Attribution

Proteins: Structure, Function, and Bioinformatics, v. 64, issue 2, p. 486-501