Event Title

Protective effect of Malate and Lactate Dehydrogenase on 6DHNAD

Mentor 1

Prof. Graham Moran

Location

Union Wisconsin Room

Start Date

29-4-2016 1:30 PM

End Date

29-4-2016 3:30 PM

Description

The Moran lab recently discovered a new cellular house-keeping enzyme of some importance. This enzyme, renalase, oxidizes isomers of NADH and NADPH that are inhibitory to primary metabolism. As part of the effort to show the influence of these isomers on primary metabolism the Moran group in collaboration with the group of Audrey Lamb at the University of Kansas have solved structures of lactate dehydrogenase (LDH) and malate dehydrogenase (MDH) with one of the inhibitory isomers bound in the active site. The difficulty is that the inhibitor is only quazi stable. It is therefore necessary to demonstrate that the inhibitory isomer is bound and not some other isosteric NAD molecule derived from decomposition. This project will attempt to prove that the 6DHNAD isomer is bound in the active site of the enzymes whose structures solved by X-ray crystallography. We used absorption spectroscopy to find the stability of 6DHNAD with and without MDH and LDH. 6DHNAD has 2 chromophoric moieties; the adenine base and the dihydronicotinamide base. When the later decomposes, absorption at 340 nm is lost. As such we use time-dependent absorption spectroscopy to determine how fast it degrades when in presence of MDH and LDH. The experiments are still in progress- we found that the 6DHNAD under crystallography conditions does not degrade as rapidly as it does in normal buffers. This is a good initial indication that crystallographic structure does have 6DHNAD in its active site. This is a very important addition to a research paper that is currently being written. These data will very what was seen in the crystal structure and allow us to publish these structures as inhibited complexes. This will form part of a study in which we show the metabolic impact of renalase.

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Apr 29th, 1:30 PM Apr 29th, 3:30 PM

Protective effect of Malate and Lactate Dehydrogenase on 6DHNAD

Union Wisconsin Room

The Moran lab recently discovered a new cellular house-keeping enzyme of some importance. This enzyme, renalase, oxidizes isomers of NADH and NADPH that are inhibitory to primary metabolism. As part of the effort to show the influence of these isomers on primary metabolism the Moran group in collaboration with the group of Audrey Lamb at the University of Kansas have solved structures of lactate dehydrogenase (LDH) and malate dehydrogenase (MDH) with one of the inhibitory isomers bound in the active site. The difficulty is that the inhibitor is only quazi stable. It is therefore necessary to demonstrate that the inhibitory isomer is bound and not some other isosteric NAD molecule derived from decomposition. This project will attempt to prove that the 6DHNAD isomer is bound in the active site of the enzymes whose structures solved by X-ray crystallography. We used absorption spectroscopy to find the stability of 6DHNAD with and without MDH and LDH. 6DHNAD has 2 chromophoric moieties; the adenine base and the dihydronicotinamide base. When the later decomposes, absorption at 340 nm is lost. As such we use time-dependent absorption spectroscopy to determine how fast it degrades when in presence of MDH and LDH. The experiments are still in progress- we found that the 6DHNAD under crystallography conditions does not degrade as rapidly as it does in normal buffers. This is a good initial indication that crystallographic structure does have 6DHNAD in its active site. This is a very important addition to a research paper that is currently being written. These data will very what was seen in the crystal structure and allow us to publish these structures as inhibited complexes. This will form part of a study in which we show the metabolic impact of renalase.