title: Catalytic Role of the Substrate Defines Specificity of Therapeutic l-Asparaginase.
abstract: Type II bacterial L-asparaginases (L-ASP) have played an important therapeutic role in cancer treatment for over four decades, yet their exact reaction mechanism remains elusive. L-ASP from Escherichia coli deamidates asparagine (Asn) and glutamine, with an ~10(4) higher specificity (kcat/Km) for asparagine despite only one methylene difference in length. Through a sensitive kinetic approach, we quantify competition among the substrates and interpret its clinical role. To understand specificity, we use molecular simulations to characterize enzyme interactions with substrates and a product (aspartate). We present evidence that the aspartate product in the crystal structure of L-ASP exists in an unusual α-COOH protonation state. Consequently, the set of enzyme-product interactions found in the crystal structure, which guided prior mechanistic interpretations, differs from those observed in dynamic simulations of the enzyme with the substrates. Finally, we probe the initial nucleophilic attack with ab initio simulations. The unusual protonation state reappears, suggesting that crystal structures (wild type and a T89V mutant) represent intermediate steps rather than initial binding. Also, a proton transfers spontaneously to Asn, advancing a new hypothesis that the substrate's α-carboxyl serves as a proton acceptor and activates one of the catalytic threonines during L-ASP's nucleophilic attack on the amide carbon. That hypothesis explains for the first time why proximity of the substrate α-COO(-) group to the carboxamide is absolutely required for catalysis. The substrate's catalytic role is likely the determining factor in enzyme specificity as it constrains the allowed distance between the backbone carboxyl and the amide carbon of any L-ASP substrate.
authors.affiliation: Department of Biology, University of Maryland, College Park, MD 20742, USA.
authors.forename: Andriy
authors.initials: A
authors.lastname: Anishkin
authors.affiliation: Center for Biological and Engineering Sciences, Sandia National Laboratories, Albuquerque, NM 87185-1315, USA.
authors.forename: Juan M
authors.initials: JM
authors.lastname: Vanegas
authors.affiliation: Center for Biological and Engineering Sciences, Sandia National Laboratories, Albuquerque, NM 87185-1315, USA.
authors.forename: David M
authors.initials: DM
authors.lastname: Rogers
authors.affiliation: Department of Bioinformatics and Computational Biology and Department of Systems Biology, University of Texas MD Anderson Cancer Center, Houston, TX 77230-1402, USA.
authors.forename: Philip L
authors.initials: PL
authors.lastname: Lorenzi
authors.affiliation: Department of Bioinformatics and Computational Biology and Department of Systems Biology, University of Texas MD Anderson Cancer Center, Houston, TX 77230-1402, USA.
authors.forename: Wai Kin
authors.initials: WK
authors.lastname: Chan
authors.affiliation: Department of Bioinformatics and Computational Biology and Department of Systems Biology, University of Texas MD Anderson Cancer Center, Houston, TX 77230-1402, USA.
authors.forename: Preeti
authors.initials: P
authors.lastname: Purwaha
authors.affiliation: Department of Bioinformatics and Computational Biology and Department of Systems Biology, University of Texas MD Anderson Cancer Center, Houston, TX 77230-1402, USA.
authors.forename: John N
authors.initials: JN
authors.lastname: Weinstein
authors.affiliation: Department of Biology, University of Maryland, College Park, MD 20742, USA. Electronic address: sukharev@umd.edu.
authors.forename: Sergei
authors.initials: S
authors.lastname: Sukharev
authors.affiliation: Center for Biological and Engineering Sciences, Sandia National Laboratories, Albuquerque, NM 87185-1315, USA. Electronic address: slrempe@sandia.gov.
authors.forename: Susan B
authors.initials: SB
authors.lastname: Rempe
mesh_terms: D001215:Asparaginase / Q000378:metabolism* / Q000648:ultrastructure*; D001216:Asparagine / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D002384:Catalysis; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000201:enzymology*; D005973:Glutamine / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation
chemical_list: D005973:Glutamine; D001216:Asparagine; D001215:Asparaginase
keywords: ab initio simulations; enzyme catalysis; enzyme kinetics; mass spectrometry (MS); molecular dynamics