PMID: 9552159Apr 29, 1998Paper

Engineering of a stable mutant malic enzyme by introducing an extra ion-pair to the protein

Proteins
S M HuangG G Chang

Abstract

A double mutant (R9E/M17K) of pigeon liver malic enzyme with glutamate and lysine replaced for arginine and methionine at positions 9 and 17, respectively, was found to be much more stable in urea and thermal denaturation, but was enzymatically less active than the wild-type enzyme (WT). Unfolding of the enzyme by urea produced a large red shifting of the protein fluorescence maximum from 320 to 360 nm, which was completely reversible upon dilution. Analysis of the denaturation curves monitored by enzyme activity lost suggested that a putative intermediate was involved in the denaturation process. The half unfolding urea concentration, measured by fluorescence spectral changes, increased from 2.24 M for WT to 3.13 M for R9E/M17K. The melting temperature increased by approximately 10 degrees C for R9E/M17K compared with that for WT. Kinetic analysis of the thermal inactivation at 58 degrees C also conformed to a three-state model with the rate constant for the intermediate state of R9E/M17K (k2 = 0.03 min(-1)) being much smaller than the WT value (k2 = 2.39 min(-1)). Results obtained from single mutants indicated that the decreasing enzyme activity of R9E/M17K was exclusively due to R9 mutation, which increased the K(mMn) and K(...Continue Reading

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Citations

Jan 16, 2002·Protein Science : a Publication of the Protein Society·Zhiru YangLiang Tong
Sep 4, 2013·Applied Biochemistry and Biotechnology·Hossein Zarei JalianiSaeed Talebi
Feb 13, 2007·International Journal of Biological Macromolecules·Claudia P SpampinatoCarlos S Andreo

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