Metal and pH dependence of heptapeptide catalysis by human matrilysin

Biochemistry
J ChaD S Auld

Abstract

Human matrilysin devoid of its propeptide is expressed in Escherichia coli and purified to homogeneity by heparin chromatography after refolding of the guanidine hydrochloride solubilized protein. Matrilysin autolytically removes its N-terminal tripeptide Met-Tyr-Ser during the refolding process. The enzyme contains 1.91 +/- 0.08 zinc atoms/mol of protein and retains full activity when stored several months at 4 degrees C. It hydrolyzes the fluorescent substrate Dns-PLALWAR at the Ala-Leu bond with a kcat of 3.1 s-1 and K(m) of 1.8 x 10(-5) M at pH 7.5, 37 degrees C, values closely similar to those for the matrilysin produced by activation of the Chinese hamster ovary and E. coli-expressed promatrilysin. The properties of this form of matrilysin demonstrate that the propeptide is not essential for proper folding or stability of the enzyme but likely determines the N-terminal amino acid of the mature enzyme. The pH dependence of kcat/K(m) for Dns-PLALWAR shows that matrilysin has a broad pH optimum (5.0-9.0) and the pKa values obtained are 4.3 and 9.6 at 25 degrees C. The activity is inhibited by several metal binding agents including 1, 10-phenanthroline, OP, but not by the nonchelating isomer, 1,7-phenanthroline. OP inhibits i...Continue Reading

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Citations

Feb 3, 2009·The Journal of Biological Chemistry·Carole L WilsonWilliam C Parks
Nov 20, 2002·Biological Chemistry·Bruce A DollJudith S Bond
Aug 9, 2011·Bioscience, Biotechnology, and Biochemistry·Tomoko MiyakeKuniyo Inouye
May 3, 2012·Langmuir : the ACS Journal of Surfaces and Colloids·Amanda J Brindley, Rachel W Martin
Jan 9, 2008·ChemMedChem·Arpita AgrawalSeth M Cohen
Nov 23, 2010·The FEBS Journal·Elin Hadler-OlsenJan-Olof Winberg
May 27, 2008·Biophysical Journal·Enrico Di StasioRaimondo De Cristofaro
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May 28, 2016·Journal of Biological Inorganic Chemistry : JBIC : a Publication of the Society of Biological Inorganic Chemistry·Fan MengMichael W Crowder
Nov 16, 2005·The Journal of Biological Chemistry·Patricia J AndersonJ Evan Sadler
Jan 4, 2015·The Journal of Biological Chemistry·Mar López-PelegrínF Xavier Gomis-Rüth
Jul 7, 2021·Journal of Biological Inorganic Chemistry : JBIC : a Publication of the Society of Biological Inorganic Chemistry·Ann VargheseTatyana G Karabencheva-Christova
May 29, 2015·Biochemistry·Hao YangDavid L Tierney
May 19, 2018·Chemical Reviews·Joan L ArolasF Xavier Gomis-Rüth
Apr 27, 2017·Biochimica Et Biophysica Acta. Molecular Cell Research·Steven R Van DorenYan G Fulcher

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