17 Aug Question: The Enzyme Formate Dehydrogenase From Moorella Hydrogenicales, Can Catalyze The Oxidation Of Formate To CO2, Using The NAD+ As An Electron Acceptor. (See Chapter 13.4 For More On Biological Redox Reactions.) This Reaction Can Be Written Thusly: HCO2– + NAD+ ® CO2 + NADH In Order To Obtain Insight Into The Mechanism Of This Kinase, You Perform A Kinetic …
The enzyme formate dehydrogenase from Moorella hydrogenicales, can catalyze the oxidation of formate to CO2, using the NAD+ as an electron acceptor. (See Chapter 13.4 for more on biological redox reactions.) This reaction can be written thusly:
HCO2– + NAD+ ® CO2 + NADH
In order to obtain insight into the mechanism of this kinase, you perform a kinetic analysis with varying amounts of formate and NAD+. Each reaction contains 4 nM formate dehydrogenase.
| [HCO2–]
(mM) |
initial velocity (nM/s) when [NAD+] (µM) = | ||||
| 20 | 60 | 200 | 600 | 2000 | |
| 0.1 | 3.7 | 10 | 24 | 43 | 60 |
| 0.3 | 10 | 28 | 68 | 120 | 158 |
| 1 | 28 | 77 | 174 | 275 | 365 |
| 3 | 61 | 153 | 341 | 509 | 611 |
| 10 | 91 | 227 | 474 | 640 | 795 |
With which of these mechanisms are most consistent with these data for this enzyme?
a) “Ping-Pong” mechanism
b) ternary complex with ordered pathway (which one binds first?)
c) ternary complex with random pathway
Briefly justify your conclusion.
Determine the following parameters for formate dehydrogenase from M. hydrogenicales:
Km and/or Kd for formate
Km and/or Kd for NAD+
kcat of the enzyme
Note: For full credit on this problem, you must show all work. If you used an Excel spreadsheet, you must attach this, and it must be clearly annotated. (i.e. it should say what each calculation is doing, what the units are for each associated number, etc.)
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