Lineweaver-Burk Plot: Calculate Km Value Easily

The Lineweaver-Burk plot, a graphical representation derived from the Michaelis-Menten equation, serves as an invaluable tool in enzyme kinetics for determining critical parameters. Enzyme kinetics studies carried out in labs provide the raw data needed to construct these plots. Km, or the Michaelis constant, represents the substrate concentration at which the reaction rate is half of Vmax, the maximum reaction rate. Researchers at institutions often use specialized software, such as GraphPad Prism, to analyze enzymatic reactions, making it easier to plot data. The plot itself is essentially a double reciprocal graph, plotting 1/V (the inverse of velocity) against 1/[S] (the inverse of substrate concentration), allowing scientists to understand how to calculate Km from a Lineweaver-Burk plot and Vmax, providing insights into enzyme behavior and reaction mechanisms.

Enzyme kinetics is the study of the rates of enzyme-catalyzed reactions.

It's a cornerstone of biochemistry, providing insights into how enzymes function, interact with substrates and inhibitors, and ultimately, how they orchestrate the myriad biochemical processes essential for life. Understanding enzyme kinetics allows us to decipher the mechanisms of enzymatic reactions and predict how changes in conditions affect these reactions.

This understanding is critical for rational drug design, metabolic engineering, and diagnostics.

Why Study Enzyme Kinetics?

Enzymes are biological catalysts that accelerate chemical reactions within living organisms. Understanding the speed or rate at which they do this, and the factors that influence that rate, is fundamental to grasping biological processes.

Enzyme kinetics gives us tools to quantitatively describe enzyme activity and its responsiveness to changes in the environment. This quantitative description is vital for creating models to explain and predict biological behaviour.

Key Concepts in Enzyme Kinetics

Several factors influence the rate of enzyme-catalyzed reactions.

Reaction Rates

The reaction rate measures how quickly a substrate is converted into a product by an enzyme. Understanding reaction rates helps us quantify enzyme efficiency and predict how changes in reaction conditions (substrate concentration, enzyme concentration) can impact product formation.

Factors Affecting Reaction Rates

Several factors affect reaction rates:

• Temperature: Enzyme activity typically increases with temperature, up to a certain point. Beyond this optimal temperature, the enzyme can denature and lose activity.

• pH: Enzymes have an optimal pH range. Deviations from this range can disrupt the enzyme's structure and catalytic activity.

• Enzyme Concentration: Generally, increasing enzyme concentration increases the reaction rate, assuming substrate is in excess.

• Substrate Concentration: The reaction rate typically increases with substrate concentration until the enzyme becomes saturated. At saturation, all enzyme active sites are occupied, and the reaction rate reaches its maximum.

• Inhibitors: Inhibitors are molecules that decrease enzyme activity. They can be competitive (competing with the substrate for the active site) or non-competitive (binding elsewhere on the enzyme and altering its shape).

The Significance of Enzyme Kinetics in Drug Development and Metabolic Pathway Analysis

Enzyme kinetics plays a pivotal role in modern drug development.

Many drugs function by inhibiting specific enzymes involved in disease pathways. Understanding the kinetics of enzyme inhibition is crucial for designing effective drugs. Researchers use enzyme kinetics to optimize drug candidates, ensuring they bind strongly to their target enzyme and effectively reduce its activity.

Enzyme kinetics also provides a framework for studying metabolic pathways.

By analyzing the kinetic properties of enzymes within a pathway, scientists can understand how metabolites flow through the network and how changes in enzyme activity affect the overall flux. This knowledge is essential for metabolic engineering, where researchers aim to optimize metabolic pathways to enhance the production of desired compounds or to correct metabolic disorders.

Michaelis-Menten Kinetics: Unveiling the Enzyme-Substrate Relationship

Enzyme kinetics is the study of the rates of enzyme-catalyzed reactions. It's a cornerstone of biochemistry, providing insights into how enzymes function, interact with substrates and inhibitors, and ultimately, how they orchestrate the myriad biochemical processes essential for life. Understanding enzyme kinetics allows us to decipher the mechani...

To truly understand enzyme behavior, we delve into the realm of Michaelis-Menten kinetics. This model provides a fundamental framework for understanding the relationship between enzyme, substrate, and reaction velocity. It reveals how enzymes bind to substrates and how this interaction dictates the speed at which a reaction proceeds. However, understanding the assumptions and limitations of the Michaelis-Menten model is as crucial as understanding the equation itself.

The Foundation: Assumptions of Michaelis-Menten Kinetics

The Michaelis-Menten model is built upon several key assumptions.

Firstly, it assumes the formation of an enzyme-substrate complex (ES) as an essential intermediate step.

Secondly, the steady-state assumption is crucial. This assumes that the concentration of the ES complex remains relatively constant over time. The rate of ES complex formation equals the rate of its breakdown.

Thirdly, the model typically considers the reaction in its initial stages, where the concentration of the product is negligible. This ensures that the reverse reaction (product back to substrate) is insignificant.

Finally, the enzyme concentration must be much lower than the substrate concentration.

Decoding the Michaelis-Menten Equation

The heart of Michaelis-Menten kinetics lies in its defining equation:

v = (Vmax * [S]) / (Km + [S])

Where:

• v represents the initial reaction velocity.
• Vmax represents the maximum velocity of the reaction.
• [S] represents the substrate concentration.
• Km represents the Michaelis constant.

Let's break down each component to fully grasp its significance.

Understanding Maximum Velocity (Vmax)

Vmax is the theoretical maximum rate of the reaction. It is achieved when the enzyme is fully saturated with the substrate. Essentially, every enzyme molecule is actively converting substrate to product, and increasing the substrate concentration further will not increase the reaction rate. Vmax is directly proportional to the enzyme concentration; double the enzyme concentration, and you double the Vmax.

The Significance of the Michaelis Constant (Km)

Km is a crucial parameter that reflects the affinity of the enzyme for its substrate. It's defined as the substrate concentration ([S]) at which the reaction rate (v) is half of Vmax.

A low Km indicates a high affinity: the enzyme binds strongly to the substrate, and only a small amount of substrate is needed to reach half of the maximum velocity.

Conversely, a high Km indicates a low affinity: the enzyme binds weakly to the substrate, and a larger amount of substrate is required to achieve half of Vmax. Km is an intrinsic property of the enzyme-substrate pair and is independent of enzyme concentration.

Substrate Concentration ([S]) and Reaction Velocity

The substrate concentration, denoted as [S], plays a direct role in determining the reaction velocity (v).

At low substrate concentrations ([S] << Km), the reaction velocity increases almost linearly with increasing [S]. The enzyme has plenty of active sites available, and the rate is limited by the availability of the substrate.

As [S] increases, the reaction velocity starts to level off, approaching Vmax. The enzyme active sites are becoming increasingly saturated with substrate.

At high substrate concentrations ([S] >> Km), the reaction velocity approaches Vmax. The enzyme is saturated, and the reaction rate is limited by the enzyme's catalytic turnover rate.

The Importance of Initial Velocity (vo or Vi)

In enzyme kinetics, we primarily focus on the initial velocity (vo or Vi) of the reaction. This is the reaction rate measured at the very beginning of the reaction, when the product concentration is negligible.

Measuring initial velocity ensures that we are only considering the forward reaction. We avoid complications arising from product inhibition or the reverse reaction.

Initial velocity is determined by measuring the change in product concentration (or substrate concentration) over a short period at the beginning of the reaction.

Limitations of the Direct Michaelis-Menten Plot

While the Michaelis-Menten plot (velocity vs. substrate concentration) provides a visual representation of enzyme kinetics, directly determining Km and Vmax from the curve can be challenging.

Accurately determining Vmax from the Michaelis-Menten plot is difficult because the curve approaches Vmax asymptotically. It's often hard to pinpoint the exact value at which the reaction reaches its maximum rate.

Similarly, determining Km, which is the substrate concentration at Vmax/2, also relies on accurately knowing Vmax. Uncertainty in Vmax translates into uncertainty in Km.

At high substrate concentrations, small errors in velocity measurements can have a significant impact on the estimation of Vmax and Km. The curve becomes relatively flat, making it hard to distinguish between different velocity values. These limitations led to the development of alternative graphical methods, such as the Lineweaver-Burk plot, which we will explore further.

Leonor Michaelis and Maud Menten: Pioneers of Enzyme Kinetics

Leonor Michaelis (1875-1949) and Maud Menten (1879-1960) were the brilliant minds behind the Michaelis-Menten equation. Their groundbreaking work in the early 20th century revolutionized our understanding of enzyme kinetics.

Michaelis, a German biochemist, made significant contributions to various fields, including histology and bacteriology.

Menten, a Canadian physician and biochemist, made important contributions to medicine and biochemistry.

Their collaboration led to the publication of their seminal paper in 1913, which laid the foundation for modern enzyme kinetics. The Michaelis-Menten equation and their model remain fundamental tools in biochemical research, a testament to their lasting legacy.

The Lineweaver-Burk Plot: A Linear Transformation for Clarity

Michaelis-Menten kinetics provides a fundamental framework for understanding enzyme behavior. However, the hyperbolic nature of the Michaelis-Menten plot can make precise determination of kinetic parameters, particularly Vmax and Km, challenging. To address this, the Lineweaver-Burk plot offers a powerful alternative: a linear transformation that simplifies the analysis and interpretation of enzyme kinetic data.

Introducing the Double Reciprocal Plot

The Lineweaver-Burk plot, also known as the double reciprocal plot, is a graphical representation of the Lineweaver-Burk equation:

1/v = (Km/Vmax) (1/[S]) + 1/Vmax*

This equation is derived by taking the reciprocal of both sides of the Michaelis-Menten equation.

The "double reciprocal" designation arises from the fact that the plot graphs the reciprocal of the reaction velocity (1/v) against the reciprocal of the substrate concentration (1/[S]).

This transformation results in a linear relationship, making it easier to determine the kinetic parameters Vmax and Km.

Overcoming the Limitations of the Michaelis-Menten Plot

The primary advantage of the Lineweaver-Burk plot is its ability to linearize the relationship between substrate concentration and reaction velocity.

In the Michaelis-Menten plot, accurately determining Vmax can be difficult because it requires extrapolating the curve to infinite substrate concentration.

The Lineweaver-Burk plot transforms the hyperbolic Michaelis-Menten curve into a straight line, allowing for a more precise determination of Vmax and Km from the intercepts of the line.

This linearization simplifies the analysis and reduces the uncertainty in parameter estimation.

Key Features and Their Significance

The Lineweaver-Burk plot provides a clear visual representation of enzyme kinetics, with each feature of the plot holding significant meaning:

Y-intercept: Determining Vmax

The y-intercept of the Lineweaver-Burk plot represents 1/Vmax.

Therefore, the Vmax can be easily calculated by taking the reciprocal of the y-intercept value.

This provides a more straightforward and accurate method for determining the maximum reaction velocity compared to the Michaelis-Menten plot.

X-intercept: Finding Km

The x-intercept of the Lineweaver-Burk plot represents -1/Km.

Consequently, the Km value can be obtained by taking the negative reciprocal of the x-intercept value.

The Km (Michaelis constant) indicates the substrate concentration at which the reaction rate is half of Vmax, reflecting the enzyme's affinity for its substrate.

Slope: Unveiling the Relationship Between Km and Vmax

The slope of the Lineweaver-Burk plot is equal to Km/Vmax.

This relationship provides additional insight into the kinetic parameters and can be used to validate the values obtained from the intercepts.

Changes in the slope, along with changes in the intercepts, can indicate different mechanisms of enzyme inhibition.

Lineweaver and Burk: The Pioneers of Linear Transformation

The Lineweaver-Burk plot is named after Hans Lineweaver and Dean Burk, who introduced this graphical method in 1934.

Hans Lineweaver was an American biochemist known for his work on enzyme kinetics and food chemistry.

Dean Burk was an American biochemist and cancer researcher.

Their collaboration resulted in a widely used tool that has significantly advanced our understanding of enzyme behavior.

Creating and Interpreting Lineweaver-Burk Plots: A Step-by-Step Guide

Michaelis-Menten kinetics provides a fundamental framework for understanding enzyme behavior. However, the hyperbolic nature of the Michaelis-Menten plot can make precise determination of kinetic parameters, particularly Vmax and Km, challenging. To address this, the Lineweaver-Burk plot offers a valuable alternative, transforming the hyperbolic relationship into a linear one. This section provides a practical, step-by-step guide to generating and analyzing Lineweaver-Burk plots, enabling you to extract meaningful insights from your enzyme kinetics experiments.

Obtaining Enzyme Kinetic Data: The Foundation of Your Plot

The first step in creating a Lineweaver-Burk plot is to acquire accurate and reliable enzyme kinetic data. This typically involves measuring the initial reaction rates (v0) at various substrate concentrations ([S]). Here's what you need to do:

1. Prepare Your Enzyme and Substrate Solutions: Ensure both enzyme and substrate are of high purity and accurately quantified.

2. Set Up Your Reactions: Prepare a series of reaction mixtures with varying substrate concentrations, keeping the enzyme concentration constant. It is crucial that you cover a broad range of substrate concentrations, ideally including concentrations significantly below and above the expected Km value.

3. Initiate the Reaction: Start the reaction by adding the enzyme to the substrate solution. Ensure rapid mixing to guarantee a homogenous reaction environment.

4. Measure Initial Velocities (v0): Measure the rate of product formation or substrate consumption over time. It's vital to measure the initial velocity (v0) – the rate at the very beginning of the reaction when substrate depletion is negligible and the reverse reaction is insignificant. Collect multiple data points during the initial linear phase of the reaction.

The Role of Spectrophotometry

Spectrophotometry is a common and powerful technique for measuring reaction rates in enzyme kinetics. Many enzymatic reactions involve a change in absorbance at a specific wavelength, either due to the formation of a colored product or the consumption of a substrate that absorbs light.

By monitoring the change in absorbance over time, you can determine the reaction rate. This method offers high sensitivity and ease of use, making it a staple in enzyme kinetics experiments.

Transforming Data to Reciprocal Values

The Lineweaver-Burk plot is based on the double reciprocal of the Michaelis-Menten equation. This means you need to transform your experimental data by taking the reciprocal of both the substrate concentrations ([S]) and the initial velocities (v0).

Understanding Reciprocals

A reciprocal is simply 1 divided by a number. For example, the reciprocal of 5 is 1/5 = 0.2. The reciprocal transformation is what linearizes the Michaelis-Menten equation, making it easier to analyze graphically.

To prepare your data:

1. Calculate 1/[S] for each substrate concentration.

2. Calculate 1/v0 for each corresponding initial velocity.

Now you have the data points needed to create your Lineweaver-Burk plot.

Creating the Lineweaver-Burk Plot

With your transformed data, you're ready to create the Lineweaver-Burk plot. This is best done using graphing software such as GraphPad Prism, Origin, or even Microsoft Excel.

1. Plot the Data: Plot 1/v0 on the y-axis and 1/[S] on the x-axis. Each data point represents a pair of reciprocal values obtained from your experiment.

2. Add a Trendline: Use the graphing software to add a linear trendline (also known as a regression line) to your data points. The goal is to find the line that best fits the data.

3. Display the Equation: Most graphing software can display the equation of the trendline on the graph. This equation will be in the form y = mx + c, where m is the slope and c is the y-intercept.

Determining Km and Vmax Using Linear Regression

Linear regression is a statistical method used to find the best-fitting straight line for a set of data points. In the context of the Lineweaver-Burk plot, it allows us to accurately determine the slope and intercepts of the line, which are directly related to Km and Vmax.

1. Determine the Y-Intercept: The y-intercept of the Lineweaver-Burk plot represents 1/Vmax. Therefore, Vmax is simply the reciprocal of the y-intercept. Vmax = 1/y-intercept.

2. Determine the X-Intercept: The x-intercept of the Lineweaver-Burk plot represents -1/Km. Therefore, Km is the negative reciprocal of the x-intercept. Km = -1/x-intercept.

3. Calculate the Slope: The slope of the line is equal to Km/Vmax. You can use this to double-check your Km and Vmax values, especially if you are concerned about the accuracy of the intercepts.

By accurately determining the slope and intercepts of the Lineweaver-Burk plot using linear regression, you can obtain reliable estimates of Km and Vmax for your enzyme.

Statistical Validation with Data Analysis Software

While the Lineweaver-Burk plot provides a visual and straightforward method for estimating Km and Vmax, it's essential to validate your results using statistical data analysis software. Programs like GraphPad Prism or R can perform more rigorous regression analysis.

This is important for several reasons:

• Error Estimation: Data analysis software provides estimates of the standard errors associated with Km and Vmax, giving you a sense of the uncertainty in your measurements.

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• Goodness-of-Fit: Software provides measures of goodness-of-fit, such as the R-squared value, which tells you how well the linear model fits your data. A low R-squared value may indicate that a linear model is not appropriate or that there are significant errors in your data.

• Outlier Detection: Statistical software can help identify outlier data points that may be skewing your results.

By using data analysis software, you can increase the confidence in your Km and Vmax estimates and ensure that your conclusions are statistically sound.

Applications of the Lineweaver-Burk Plot: From Enzyme Characterization to Inhibition Studies

Michaelis-Menten kinetics provides a fundamental framework for understanding enzyme behavior. However, the hyperbolic nature of the Michaelis-Menten plot can make precise determination of kinetic parameters, particularly Vmax and Km, challenging. To address this, the Lineweaver-Burk plot offers a valuable, albeit not without limitations, alternative for visualizing and analyzing enzyme kinetics data. This section delves into the practical applications of the Lineweaver-Burk plot, focusing on enzyme characterization, inhibition studies, and comparative enzyme efficiency analysis.

Determining Kinetic Parameters (Km and Vmax)

The Lineweaver-Burk plot provides a straightforward method for determining the critical kinetic parameters of an enzyme: Km and Vmax. As a double reciprocal plot, it transforms the hyperbolic Michaelis-Menten curve into a linear relationship.

The y-intercept of the Lineweaver-Burk plot represents 1/Vmax, allowing for easy calculation of the maximum reaction velocity. Similarly, the x-intercept corresponds to -1/Km, providing a direct means of determining the Michaelis constant, which reflects the enzyme's affinity for its substrate.

By fitting a straight line to the experimental data points and extrapolating to the intercepts, researchers can quickly estimate these parameters. Keep in mind the limitations previously discussed.

Studying Enzyme Inhibition

The Lineweaver-Burk plot shines when investigating enzyme inhibition. Different types of inhibitors exert distinct effects on the plot, enabling researchers to identify the mode of inhibition and quantify the inhibitor's potency. Let's explore the common types.

Competitive Inhibition

In competitive inhibition, the inhibitor binds to the active site of the enzyme, competing with the substrate.

This type of inhibition increases the apparent Km (decreases the x-intercept) while leaving Vmax unchanged (y-intercept remains the same). The Lineweaver-Burk plot shows a family of lines intersecting on the y-axis.

Non-Competitive Inhibition

Non-competitive inhibitors bind to a site on the enzyme distinct from the active site, altering the enzyme's conformation and reducing its catalytic efficiency.

Vmax is decreased (y-intercept increases), while Km remains the same (x-intercept stays constant). On the Lineweaver-Burk plot, the lines intersect on the x-axis.

Uncompetitive Inhibition

Uncompetitive inhibitors bind only to the enzyme-substrate complex, preventing the reaction from proceeding.

Both Km and Vmax are decreased proportionally. Consequently, the Lineweaver-Burk plot displays a series of parallel lines.

Mixed Inhibition

Mixed inhibition is a combination of competitive and non-competitive inhibition.

The inhibitor can bind to either the free enzyme or the enzyme-substrate complex. Both Km and Vmax are affected.

By carefully analyzing the changes in the intercepts and slope of the Lineweaver-Burk plot in the presence of different inhibitors, researchers can gain valuable insights into the mechanisms of enzyme inhibition.

Comparing Enzyme Efficiency

The Lineweaver-Burk plot can be used to compare the catalytic efficiency of different enzymes. By determining the Km and Vmax values for each enzyme, you can calculate the catalytic efficiency (Vmax/Km).

A higher catalytic efficiency indicates a more efficient enzyme. You can graphically compare the slopes of the Lineweaver-Burk plots for different enzymes acting on the same substrate. The enzyme with the steeper slope has a lower catalytic efficiency, and vice versa. This allows for a visual comparison of enzyme performance.

Advantages and Limitations: A Balanced Perspective on the Lineweaver-Burk Plot

Applications of the Lineweaver-Burk Plot: From Enzyme Characterization to Inhibition Studies Michaelis-Menten kinetics provides a fundamental framework for understanding enzyme behavior. However, the hyperbolic nature of the Michaelis-Menten plot can make precise determination of kinetic parameters, particularly Vmax and Km, challenging. To address this, the Lineweaver-Burk plot offers a linear transformation that simplifies analysis. But like any scientific tool, it's crucial to understand both its strengths and weaknesses.

The Allure of Linearity: Advantages of the Lineweaver-Burk Plot

The primary advantage of the Lineweaver-Burk plot lies in its linear representation of enzyme kinetics data. This linearity simplifies the determination of key kinetic parameters, Km and Vmax, which are otherwise difficult to ascertain precisely from the hyperbolic Michaelis-Menten plot.

Simplified Parameter Determination

By plotting the reciprocal of the reaction rate (1/v) against the reciprocal of the substrate concentration (1/[S]), a straight line is obtained. This allows for a straightforward determination of Vmax from the y-intercept (1/Vmax) and Km from the x-intercept (-1/Km). This visual clarity is particularly helpful for educational purposes and quick estimations.

Visualizing Enzyme Inhibition

The Lineweaver-Burk plot is also invaluable for visualizing the effects of different types of enzyme inhibition. Competitive, non-competitive, and uncompetitive inhibitors each produce characteristic changes in the slope and intercepts of the plot, allowing for easy identification of the mode of inhibition.

This visual distinction is a powerful tool for understanding how different inhibitors interact with enzymes and affect their activity. Researchers can quickly distinguish between different types of inhibition patterns.

Shadows of Linearity: Limitations of the Lineweaver-Burk Plot

Despite its advantages, the Lineweaver-Burk plot suffers from several limitations that can affect the accuracy of parameter estimation. It's essential to be aware of these drawbacks and to consider alternative methods when higher precision is required.

Unequal Distribution of Data and Inaccuracy at Low Substrate Concentrations

The reciprocal transformation inherent in the Lineweaver-Burk plot distorts the distribution of data points. Data points at low substrate concentrations, which are often the least accurate, are given undue weight, while points at high substrate concentrations are compressed.

This unequal weighting can lead to significant errors in the estimation of Km and Vmax, particularly when the data is noisy or when the substrate concentration range is limited.

Distortion of Experimental Error

The reciprocal transformation also distorts experimental error. Errors in measuring low reaction rates or substrate concentrations are magnified in the Lineweaver-Burk plot, while errors in measuring high values are diminished.

This can lead to an underestimation of the uncertainty in the estimated parameters and a false sense of precision.

Alternative Methods for Enhanced Accuracy

Due to the limitations of the Lineweaver-Burk plot, more sophisticated methods are now preferred for accurate parameter estimation. Non-linear regression analysis, performed directly on the Michaelis-Menten equation, provides a statistically more robust and accurate approach.

Software packages like GraphPad Prism and Origin offer non-linear regression tools that account for the error structure of the data and provide more reliable estimates of Km and Vmax. Furthermore, direct linear plots and Hanes-Woolf plots, while less commonly used, offer alternative linearizations that may be less susceptible to error distortion under specific conditions.

<h2>Frequently Asked Questions</h2>

<h3>What exactly is a Lineweaver-Burk plot used for?</h3>
A Lineweaver-Burk plot, also known as a double reciprocal plot, is a graphical representation of the Lineweaver-Burk equation. It's primarily used to analyze enzyme kinetics data, specifically to determine key parameters like the Michaelis-Menten constant (Km) and the maximum reaction rate (Vmax).

<h3>How is the Km value determined from the Lineweaver-Burk plot?</h3>
The Km value can be determined from the x-intercept of the Lineweaver-Burk plot. The x-intercept represents the value of -1/Km. Therefore, *how to calculate Km from Lineweaver Burk plot* is to take the negative reciprocal of the x-intercept.

<h3>Why is the Lineweaver-Burk plot considered a double reciprocal plot?</h3>
It's called a double reciprocal plot because both the substrate concentration ([S]) and the reaction velocity (v) are inverted. The plot graphs 1/v on the y-axis against 1/[S] on the x-axis, resulting in a linear relationship that's easier to analyze than the original Michaelis-Menten curve.

<h3>What are the limitations of using a Lineweaver-Burk plot?</h3>
While helpful, Lineweaver-Burk plots can be susceptible to inaccuracies, especially with data points at low substrate concentrations. These points have a disproportionate influence on the line, potentially leading to errors in *how to calculate Km from Lineweaver Burk plot* and Vmax. Non-linear regression methods are often preferred for more accurate results.

So, next time you're staring down a mountain of enzyme kinetics data, don't panic! Whip out the Lineweaver-Burk plot, find that x-intercept, and remember that negative reciprocal? That's right – to calculate Km from Lineweaver Burk plot, you just take the absolute value of the inverse of the x-intercept, and you're golden! Happy plotting!