(DEC 2011)
49. The hydrolysis of pyrophoshate to orthophosphate is important for several biosynthetic reactions. In E. coli, the molecular mass of the enzyme pyrophosphatase is 120 kD and it consists of six identical subunits. The enzyme activity is defined as the amount of enzyme
that hydrolyzes 10 µmol of pyrophosphate in 15 minutes at 37⁰ under standard assay condition. The purified enzyme has a V_max of 2800 units per milligram of the enzyme. How many moles of the substrate are hydrolysed per second per milligram of the enzyme when the substrate concentration is much greater than K_m?
(1) 0.05 µmol                                                           (2) 62 µmol
(3) 31.1 µmol                                                           (4) 1 µmol

The correct answer is (3) 31.1 µmol.

Introduction

Pyrophosphatase is critical for hydrolyzing pyrophosphate into orthophosphate, driving biosynthetic reactions in organisms like E. coli. Enzyme activity is often defined based on the amount of substrate converted under specific conditions, typically in units per time and per weight of enzyme. Translating this information to substrate hydrolysis rate per second per mg of enzyme, especially under saturating substrate concentration (S≫KmS≫Km), is essential for understanding catalytic efficiency and reaction kinetics. This guide walks through calculations based on the given data.

Given Data

• Molecular mass of enzyme = 120 kDa (not essential for calculation here)

• Enzyme activity defined as amount hydrolyzed: 10 µmol pyrophosphate in 15 min at 37°C

• Vmax=2800Vmax=2800 units per mg enzyme

• Substrate concentration much greater than KmKm, i.e., enzyme saturated

• 1 unit = amount of enzyme hydrolyzing 10 µmol substrate in 15 min

Step 1: Understand what a unit represents

• 1 unit = 10 µmol substrate / 15 min

• Convert to µmol per second for 1 unit:

1 unit=10 μmol15×60 s=10900=0.0111 μmol/s1 unit=15×60s10μmol=90010=0.0111μmol/s

Step 2: Calculate substrate hydrolysis per second per mg enzyme at VmaxVmax

Given activity = 2800 units/mg enzyme:

$$\text{Hydrolysis rate}=2800\times 0.0111=31.1\mu mol/\text{s}/\text{mg}$$

Interpretation

• Under saturating substrate conditions, pyrophosphatase hydrolyzes approximately 31.1 µmol pyrophosphate per second per mg enzyme.

• This rate corresponds directly to VmaxVmax, reflecting maximum catalytic capacity.

Summary Table

Conclusion

The enzyme pyrophosphatase hydrolyzes substrate at a rate of 31.1 µmol per second per milligram of enzyme when substrate concentration is saturating. This calculation translates standard activity units to a more intuitive kinetic rate, pivotal for enzyme characterization and biochemical research.

This scientific explanation supports learners and professionals in enzyme kinetics by elucidating unit conversions and catalytic rate interpretations with practical examples.The correct answer is (3) 31.1 µmol.

Title
Calculating the Substrate Hydrolysis Rate of Pyrophosphatase at Saturated Substrate Concentration

Meta Description
Learn how to convert enzyme activity units into substrate hydrolysis rates per second per milligram enzyme for pyrophosphatase. Stepwise guide using kinetic data for life science students and researchers.

Slug
calculate-substrate-hydrolysis-pyrophosphatase

Introduction
Pyrophosphatase functions to hydrolyze pyrophosphate into orthophosphate, a vital step in biosynthetic reactions. Understanding its catalytic capacity under saturating substrate concentration is important for enzyme kinetics and biochemical applications. This guide explains converting enzyme activity expressed in units into the actual substrate turnover rate in µmol per second per mg enzyme.

Given Data

• Enzyme activity definition: 10 µmol pyrophosphate hydrolyzed in 15 minutes Corresponds to 1 unit activity

• Vmax=2800Vmax=2800 units/mg enzyme

• Substrate concentration ≫Km≫Km (saturation)

Step 1: Convert one unit to substrate hydrolyzed per second

1 unit=10 μmol15×60 s=10900=0.0111 μmol/s1 unit=15×60s10μmol=90010=0.0111μmol/s

Step 2: Calculate hydrolysis rate at VmaxVmax per mg enzyme

$$\text{Rate}=2800\times 0.0111=31.1\mu mol/s/mg$$

Interpretation
Under saturated substrate concentration, pyrophosphatase hydrolyzes approximately 31.1 µmol of substrate per second per milligram of enzyme, demonstrating its maximal catalytic rate.

Summary Table

Conclusion
The calculated substrate hydrolysis rate at VmaxVmax for pyrophosphatase is 31.1 µmol/s per mg enzyme, translating standard enzyme units into a meaningful kinetic parameter. This information is critical for enzyme characterization and biochemical experiment design.