Watching an enzyme in action

An absolutely spectacular paper basically lets you watch the enzyme in action [ Proc. Natl. Acad. Sci. vol. 117 pp. 19720 – 19730 ’20 ].   Have a look if you or your institution has a subscription — but it’s probably behind a paywall — https://www.pnas.org/content/pnas/117/33/19720.full.pdf. The enzyme is 3 hydroxyanthranilate 3, 4 dioxygenase (HAO) and important member of the path from tryptophan to quinolinic acid, which ultimately is a feed stock for the crucially important redox carrier NAD+ (Nicotinamide Adenine Dinucleotide).  The reaction catalyzed is complex and involves SEVEN catalytic intermediates.  The paper shows 7 of them at a very high resolution (1.6 Angstroms).

How did they do it?  Well the study was performed at 77 Kelvin (-321 Fahrenheit) which slowed things down considerably (the intermediates were seen at 2, 3, 5, 7 and 9 MINUTES.    The reaction was not done in solution, but in a crystal of the enzyme. The crystal alone slowed the reaction rate down by 10,000 fold compared to the enzyme at the same temperature in solution.  Obviously the enzyme substrate (3 hydroxy anthanilic acid) had to diffuse into the crystal.  One would think that different parts of the crystal would achieve the correct substrate concentration at different times, making the determination of the intermediates difficult as a variety of the 7 intermediates should be present at the same time.  They used 1,000 small sized crystals of the enzyme (but they don’t say how small).

The 3 hydroxy anthranilic acid is just benzene with a carboxyl group at position 1, NH2 at position 2 and, OH at position three.  At the end the ring is opened with a carboxyl group at position 3 and CHO at position 4.   We see the substrate bind, then oxygen enter and more intermediates subsequently.  One of them is a mono-ozygenated 7 membered lactone ring

Spectacular stuff.  Among other things they used Xray crystallography to image the intermediates (using the crystals over and over, which I didn’t think possible).

 

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