[micromelissa]

A few questions regarding enzymatic kinetics.

(TLDR; forward to #1 and #2)

Here are the circumstances surrounding my assay:

1) Assay 1 has no inhibitor, 4mM ONPG substrate and 8mM b-galactosidase. Measured rate = 0.105 abs/min
2) Assay 2 has , 4mM ONPG substrate, 4mM glucose (potential inhibitor) and 8mM b-galactosidase. Measured rate - 0.106 abs/min

#1 Can I assume that Assay 2s glucose really isn't inhibiting at all because they have identical rates at identical substrate concentration?

#2 How can I deduce the structural basis for substrate specificity by examining the inhibition information? What does this inhibition tell us about how the enzyme (b-galactosidase) binds the substrate ONPG and its natural substrate (lactose)?

This was my answer:

The activity of glucose was measured at 0.105 abs/min, which is nearly identical to the value of 0.103 abs/min. The comparable assay in question had no inhibition occurring because it contained a concentration of zero galactose. Both assays were under similar experimental conditions and had an identical concentration of ONPG substrate (4mM ONPG).

Due to the similarities of the measured catalytic activity and substrate concentration, it can be concluded that glucose does not inhibit B-galactosidase. The inhibiton of B-galactosidase provides information about how the enzyme binds ONPG and the natural substrate, lactose. B-galactosidase binds ONPG and lactose in the active site. B-galactosidase binds the un-competitive galactose inhibitor in a different spot than the active site when the enzyme substrate complex is formed.

 It will bind to the E-S complex when B-galactosidase and ONPG or lactose is bound in the active site. B-galactosidase cleaves the dissacharide lactose into galatose and glucose. Galactose is then free to bind to the enzyme to inhibit the reaction when the E-S complex is formed again. Glucose is free product and will not bind to the enzyme because it is not an inhibitor of B-galactosidase.



Does my answer make sense? Am I missing something? I feel like I am missing something in the last part where I talk about the enzyme binding the substrate. I hope someone can give me a bit of feedback to point me in the right direction.


Thanks!

[Yggdrasil]

The natural substrate of β-galactosidase is lactose.  Consider the structure of lactose, and the parts of lactose the enzyme might recognize and bind.  What do your experiments with glucose tell you about which parts of lactose the enzyme recognizes?

[micromelissa]

lactose is a disaccharide consisting of a galactose and glucose molecule.

My data tells me that Galactose is an inhibitor, and glucose is not.

Therefore the enzyme should recognize glucose in the active site.

Once the lactose molecule glycosidic bond is broken, galactose is free to bind to the E-S complex and inhibit B-galactosidase for another lactose molecule.

[Yggdrasil]

Therefore the enzyme should recognize glucose in the active site.

Can you explain how you came to this conclusion?

[Babcock_Hall]

"8mM beta-galactosidase"  One comment:  This is not a realistic concentration of this enzyme in a typical enzyme assay.  One question:  Is it given in the problem that galactose is an uncompetitive inhibitor?