The computational studies will focus on two peroxidases, horse radish peroxidase-C (HRP-C), and chloroperoxidase (CPO), with the most disparate structures and functions thus far observed for this ubiquitous family of metabolizing heme proteins. One type of study will focus on the role of the heme unit itself in the formation of the common catalytically active species of the two enzymes. To this end, the electronic structure and spectra of each of the four key CPO heme species proposed to participate in its enzymatic cycle will be calculated using the semi-empirical quantum chemical method, INDO/ROHF/CI successfully used in similar past studies. A second type of study will focus on understanding how the different protein environments in the two enzymes can nevertheless lead to the formation of the same enzymatically active species. This question will be addressed by performing molecular dynamics simulations of the full precursor protein-peroxide complex of both HRP-C and CPO using the empirical energy based Amber 4.1 suite of programs. The catalytically active enzyme species will then be used to characterize enzyme-substrate complexes to identify and compare substrate binding sites and the modes of binding of typical substrates of the two enzymes. These new insights will allow continued resolution of other challenging unresolved aspects of metabolic activity. Chief among these are the identification and characterization of the molecular determinants of substrate and product specificities in the two disparate metabolic pathways observed in both enzymes.
Multidisciplinary studies are required to elucidate enzyme structure-function relationships on a molecular level. Computational biochemistry can play an important role in this effort. The computational methods to be used here are applicable to the elucidation of unresolved aspects of the function of any enzyme for which a 3D structure has been determined, either by experimental or computational techniques. It is important however that insights obtained from these computational simulations of enzyme-substrate complexes be assessed by further experimental studies. The planned collaborations will ensure that this goal is achieved. Specifically, mutations at sites predicted as important for function will be made and their effect assessed in the laboratories of Paul Ortiz de Montellano and Andrew Smith for HRP-C and of Lowell Hager for CPO. These integrated collaborative efforts between structural, computational and molecular biology should provide significantly enhanced understanding of the relationship between structure and function of these two disparate peroxidases and the origin of both similarities and differences between them. Moreover, this integrated experimental/computational approach can be generalized to structure-function studies of other enzyme families.
