Frontal affinity chromatography coupled to mass spectrometry for screening mixtures of enzyme inhibitors.
Frontal affinity chromatography coupled online to mass spectrometry (FAC/MS) has previously been used to estimate binding constants for individual protein ligands present in mixtures of compounds. In this study FAC/MS is used to determine enzyme substrate kinetic parameters and binding constants for enzyme inhibitors. Recombinant human N-acetylglucosaminyltransferase V was biotinylated and adsorbed onto immobilized streptavidin in a microcolumn (20 microL). The enzyme was shown to be catalytically competent transferring GlcNAc from the donor UDP-GlcNAc to beta-d-GlcpNAc-(1-->2)-alpha-d-Manp-(1-->6)-beta-d-Glcp-OR acceptor giving beta-d-GlcpNAc-(1-->2)-[beta-d-GlcpNAc-(1-->6)]-alpha-d-Manp-(1-->6)-beta-d-Glcp-OR as the reaction product. The kinetic parameters K(m) and V(max) for the immobilized enzyme could be determined by FAC/MS and were comparable to those measured in solution. Analysis of a mixture of eight trisaccharide analogs in a single run yielded K(d) values for each of the eight compounds ranging from 0.3 to 36 microM. These K(d) values were 2 to 10 times lower than the inhibition constants, K(I)'s, determined in solution using a standard radiochemical assay. However, the ranking order of K(d)'s was the same as the ranking of K(I) values. FAC/MS assays can therefore be employed for the rapid estimation of inhibitor K(d) values making it a valuable tool for enzyme inhibitor evaluations.
Zhang
B
B
Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2G2.
Palcic
M M
MM
Schriemer
D C
DC
Alvarez-Manilla
G
G
Pierce
M
M
Hindsgaul
O
O
D016667:Bacterial Capsules; D002846:Chromatography, Affinity / Q000379:methods*; D004791:Enzyme Inhibitors / Q000032:analysis* / Q000138:chemical synthesis / Q000494:pharmacology; D004800:Enzymes, Immobilized / Q000037:antagonists & inhibitors / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008403:Mass Screening; D013058:Mass Spectrometry / Q000379:methods*; D017351:N-Acetylglucosaminyltransferases / Q000037:antagonists & inhibitors / Q000378:metabolism; D011135:Polysaccharides, Bacterial / Q000378:metabolism*; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D014537:Uridine Diphosphate N-Acetylglucosamine / Q000378:metabolism*
D004791:Enzyme Inhibitors; D004800:Enzymes, Immobilized; D011135:Polysaccharides, Bacterial; D011994:Recombinant Proteins; C110031:capsular polysaccharide 35A; D014537:Uridine Diphosphate N-Acetylglucosamine; D017351:N-Acetylglucosaminyltransferases; C037649:N-acetyllactosaminide beta-1,6-N-acetylglucosaminyltransferase