Faculty Mentor
Dr. Elizabeth Howell
Department (e.g. History, Chemistry, Finance, etc.)
Biochemistry, Cellular, and Molecular Biology
College (e.g. College of Engineering, College of Arts & Sciences, Haslam College of Business, etc.)
College of Arts & Sciences
Year
2018
Abstract
The effect of osmolytes on ligand binding to dihydropteroate synthase
Folic Acid, or Vitamin B9, is involved in one-carbon transfer metabolism and is required for the synthesis of important amino acids and nucleotide precursors. Therefore, if there are any defects in the enzymes that are involved in its metabolism, birth defects, and cardiovascular diseases can result. Humans cannot synthesize folic acid on their own; thus, it is acquired via dietary supplements. In contrast, bacteria can synthesize folic acid. This unique feature allows drug targeting of the bacterial folate synthesis enzymes using, for example, sulfa drugs. Dihydropteroate synthase (DHPS) is one of the enzymes in the folic acid synthesis pathway in bacteria. DHPS catalyzes the reaction of 6-hydroxymethyl-7, 8-dihydropterin pyrophosphate (DHPP) with para-aminobenzoic acid (pABA) to form dihydropteroate and pyrophosphate. This is an ordered reaction with DHPP binding to the enzyme first, which then allows pABA to bind.
Folate is comprised of a pteridine ring, a pABA ring, and a glutamate tail. Previous studies in our lab have shown that osmolytes weakly interact with folate at particular atoms, which are compounds that maintain the cell’s volume and fluid balance. For example, the pteridine ring of folate prefers to interact with the osmolyte betaine while the glutamate tail of folate prefers to interact with water. When attempting to remove osmolytes from the ligand, it is more difficult to remove them compared to water molecules. This results in a weaker association of folate to the enzyme. We predict that osmolytes will weakly interact with DHPP and pABA and prevent them from associating with the DHPS enzyme. Further studies conducted in our lab have identified trehalose as an osmolyte that will preferentially interact with DHPP, whereas betaine will preferentially exclude it. We have conducted Isothermal Titration Calorimetry (ITC) and fluorescence experiments to measure the binding affinity of DHPP to DHPS. Furthermore, we have explored the effects of osmolytes and observed that trehalose weakens the association of DHPP to DHPS; whereas, betaine strengthens the association. Future studies will be conducted to understand the binding affinity of pABA using these techniques. This will be followed by studying the effect of osmolytes on pABA binding to DHPS. We predict that both trehalose and betaine will weaken the association of pABA to the enzyme.
The effect of osmolytes on ligand binding to dihydropteroate synthase
The effect of osmolytes on ligand binding to dihydropteroate synthase
Folic Acid, or Vitamin B9, is involved in one-carbon transfer metabolism and is required for the synthesis of important amino acids and nucleotide precursors. Therefore, if there are any defects in the enzymes that are involved in its metabolism, birth defects, and cardiovascular diseases can result. Humans cannot synthesize folic acid on their own; thus, it is acquired via dietary supplements. In contrast, bacteria can synthesize folic acid. This unique feature allows drug targeting of the bacterial folate synthesis enzymes using, for example, sulfa drugs. Dihydropteroate synthase (DHPS) is one of the enzymes in the folic acid synthesis pathway in bacteria. DHPS catalyzes the reaction of 6-hydroxymethyl-7, 8-dihydropterin pyrophosphate (DHPP) with para-aminobenzoic acid (pABA) to form dihydropteroate and pyrophosphate. This is an ordered reaction with DHPP binding to the enzyme first, which then allows pABA to bind.
Folate is comprised of a pteridine ring, a pABA ring, and a glutamate tail. Previous studies in our lab have shown that osmolytes weakly interact with folate at particular atoms, which are compounds that maintain the cell’s volume and fluid balance. For example, the pteridine ring of folate prefers to interact with the osmolyte betaine while the glutamate tail of folate prefers to interact with water. When attempting to remove osmolytes from the ligand, it is more difficult to remove them compared to water molecules. This results in a weaker association of folate to the enzyme. We predict that osmolytes will weakly interact with DHPP and pABA and prevent them from associating with the DHPS enzyme. Further studies conducted in our lab have identified trehalose as an osmolyte that will preferentially interact with DHPP, whereas betaine will preferentially exclude it. We have conducted Isothermal Titration Calorimetry (ITC) and fluorescence experiments to measure the binding affinity of DHPP to DHPS. Furthermore, we have explored the effects of osmolytes and observed that trehalose weakens the association of DHPP to DHPS; whereas, betaine strengthens the association. Future studies will be conducted to understand the binding affinity of pABA using these techniques. This will be followed by studying the effect of osmolytes on pABA binding to DHPS. We predict that both trehalose and betaine will weaken the association of pABA to the enzyme.