Gobind Singh

Featured Researcher:

John Caradonna & Gobind Singh Gobind Singh

Program: Beckman 2002-04
School: CAS '04
Major: Chemistry
Advisor: John Caradonna , CAS, Associate Professor,Chemistry

Inspiration: All of my life, I have been fascinated by the simple and complex occurrences in the world around me. This interest has driven me to become an active scientist. In particular, I am interested in biological science as it relates to disease and the underlying chemical basis of pathology. As a consequence of my interests in the field of chemistry and biochemistry, it was only natural for me to engage myself in a line of research dealing exactly with this particular interface of science. With my advisor and lab co-workers being role models for me both as scientists and people, I am determined more than ever to pursue a research/medical-orientated career post-graduation.

Research: I am currently investigating the mechanism of the iron-dependent enzyme, phenylalanine hydroxylase (PAH), a liver enzyme responsible for maintaining phenylalanine homeostasis in humans. Defects in the PAH gene lead to the genetic defect, phenylketonuria (PKU), the most common inborn metabolic genetic disorder of clinical significance. Approximately 1:10,000 individuals suffer from PKU in the U.S.
Work in the Caradonna laboratory characterizing two missense mutations that cause PKU showed that the mutant forms of the enzyme were more susceptible to an inactivation process than the wildtype enzyme. The inactivation is thought to be a direct consequence of irreversible specific side-chain oxidation chemistry that is induced by the nonproductive decay of an intermediate compound.

My project involves the use of liquid chromatography and mass spectroscopy techniques to investigate if any protein side chains are irreversibly modified during enzyme inactivation. A comparison of the data from wild-type PAH with inactivated PAH will allow us to determine exactly which residues are modified. This information, coupled with structural data from crystallographic studies, will allow us to locate the modification in the three dimensional structure.

In addition to understanding how the enzyme is inactivated, our laboratory is in the process of isolating and characterizing the reactive intermediate compound that is believed to be responsible for the enzyme’s overall chemistry. We are attempting to observe the presence of the intermediate iron-oxo species by using a poor substrate (para-F-phenylalanine) to slow the kinetics of the oxygen atom transfer step in the overall conversion of substrate to product. However, in order to analyze the inhibitory properties of this substrate in relation to the reactive species, it is necessary first to determine the binding affinity (Km) of para-F-Phe to PAH. We will then follow the reaction using stopped-flow rapid kinetics spectroscopic methods and attempt to identify the presence of kinetically competent intermediates.
Data from both of these studies will allow us to arrive at an understanding of the correct structure of the active site and locate the inactive site in PKU-mutant rising enzymes. With a deeper understanding of the underlying chemical defects found in PKU-inducing mutants, new approaches in the treatment of this type of disorder can hopefully follow.