Project Grant R35GM153337

TIME-RESOLVED STRUCTURAL STUDIES OF ENZYME CATALYSIS AND REGULATION - ABSTRACT ENZYME CATALYSIS IS ONE OF THE MOST IMPORTANT PHENOMENA IN BIOCHEMISTRY AND YET IS INCOMPLETELY UNDERSTOOD. NEW TIME-RESOLVED SERIAL CRYSTALLOGRAPHY METHODS NOW ALLOW ENZYME CATALYSIS TO BE OBSERVED IN REAL TIME, IN NEAR-PHYSIOLOGICAL CONDITIONS, AND AT ATOMIC RESOLUTION, ALLOWING NEW CLASSES OF EXPERIMENTS TO BE PERFORMED. THE CENTRAL GOAL OF THIS PROPOSAL IS TO BOTH DEVELOP AND USE INNOVATIVE STRUCTURAL BIOLOGY METHODS TO UNDERSTAND THE UNDERLYING PHYSICAL PRINCIPLES OF ENZYME FUNCTION. WE WILL USE TIME-RESOLVED SERIAL CRYSTALLOGRAPHY TO DETERMINE HOW CATALYSIS CHANGES ENZYME STRUCTURE AND DYNAMICS IN CYSTEINE-DEPENDENT ENZYMES. OUR INITIAL FOCUS IS ON VARIOUS ENZYMES IN THE DJ-1 SUPERFAMILY INVOLVED IN PARKINSONIAN NEURODEGENERATION, ISOCYANIDE ANTIBIOTIC DESTRUCTION, AND CARBONYL STRESS MITIGATION. A SECOND CONTRIBUTOR TO ENZYME CATALYSIS IS QUANTUM MECHANICAL EFFECTS, SOME OF WHICH ARE BECOMING ACCESSIBLE TO LARGE COMPUTATIONAL SIMULATIONS AND EXPERIMENTAL CHARACTERIZATION. WE WILL DETERMINE HOW NEGLECTED QUANTUM MECHANICAL EFFECTS AFFECT ENZYME CATALYSIS BY INCORPORATING TIME-RESOLVED CRYSTALLOGRAPHY, COMPUTATION, AND BIOCHEMISTRY TO INVESTIGATE PERVASIVE EVIDENCE OF QUANTUM MECHANICAL CHARGE TRANSFER IN CATALYSIS. THESE SCIENTIFIC GOALS WILL BE PURSUED CONCURRENTLY WITH THE DEVELOPMENT OF NEW METHODS TO EXPAND THE SCOPE AND POWER OF TIME-RESOLVED STRUCTURAL BIOLOGY EXPERIMENTS BY REDUCING BARRIERS TO PERFORMING THESE EXPERIMENTS. IN TOTAL, THIS WORK WILL USE NEW TECHNOLOGIES TO IMPROVE THE UNDERSTANDING OF FUNDAMENTAL ENZYMATIC PHENOMENA AND BROADEN THE APPLICATION OF TIME-RESOLVED STRUCTURAL BIOLOGY.