Application of mass spectrometry to determine the activity and specificity of pectin lyase A.
Electrospray ionization (ESI) with quadrupole ion-trap mass spectrometry was used to assess the activity and specificity of the enzyme pectin lyase A (PLA) (EC 4.2.2.10) on model pectins with varying degrees and patterns of methyl esterification. PLA is a pectinase which cleaves alpha-(1-->4)-glycosidic linkages in pectin by a trans-elimination process. Using pectins with different degrees and patterns of methyl esterification, there was a significant variation in the activity rate of PLA. The enzymatic products generated at various time intervals were structurally analyzed by mass spectrometry to determine the specificity of PLA. Although the preferred substrate for PLA is fully methyl esterified polygalacturonate, cleavage was also observed with a non-methyl esterified galacturonic acid residue on either the non-reducing end or the reducing end. The current study shows that although PLA prefers fully methyl esterified substrates it can also accept partially esterified ones. It also demonstrates the suitability of ESI ion-trap mass spectrometry in determining enzyme specificities.
Mutenda
Kudzai E
KE
Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230, M, Odense, Denmark.
Körner
Roman
R
Christensen
Tove M I E
TM
Mikkelsen
Jørn
J
Roepstorff
Peter
P
D002240:Carbohydrate Sequence; D008969:Molecular Sequence Data; D010368:Pectins / Q000737:chemistry / Q000378:metabolism*; D011133:Polysaccharide-Lyases / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization; D013379:Substrate Specificity
D010368:Pectins; D011133:Polysaccharide-Lyases; C027349:pectin lyase