Exploring the Biophysical Mechanisms of Taurine's Effect on Myeloperoxidase Enzymatic Kinetics in Pre-Diabetic and Type 2 Diabetic Patients

Background: investigate the enzymatic activity of Myeloperoxidase (MPO) in pre-diabetic and diabetic individuals and explore the modulation of this activity by taurine supplementation, considering its potential anti-oxidative properties and the emerging evidence of its role in glucose metabolism. Methods: This case-control study was done at the Iraqi University College of Medicine. It used advanced spectroscopic techniques and kinetic modeling to measure the amount of MPO activity in the sera of people who were healthy, pre-diabetic, and diabetic. The Lineweaver-Burk plot derived from the Michaelis-Menten equation was used to ascertain the Km and Vmax of MPO. Taurine inhibition assays were also performed to understand its effect on MPO kinetics. Results: The data showed that MPO activity increased significantly from the control group to the diabetic group. This was in line with rising HbA1c levels and BMI, suggesting a link between MPO activity, glycemic control, and obesity. The gender distribution showed no significant deviation, suggesting that the observed enzymatic and metabolic alterations are not gender biased. Conclusion: The pronounced elevation in MPO activity in diabetic individuals underscores the enzyme's potential significance in glycemic diseases. The results mean that more research needs to be done on how taurine, which is known to have anti-inflammatory and antioxidant properties, could improve MPO activity and possibly restore metabolic homeostasis, opening a new way to treat type 2 diabetes.


Introduction
Myeloperoxidase (MPO) stands out as an integral member of the peroxidase enzyme family [1].Its presence is predominantly noted within specific immune cells, namely neutrophils, monocytes, and macrophages.Beyond these immune cells, other cellular structures in our body also partake in MPO production, indicating its broader physiological significance.Diving deeper into cellular mechanisms, it's fascinating to note that MPO resides in unique compartments called azurophilic granules within these cells [2].For this purpose, the enzyme uses H₂O₂ in conjunction with halides or other compounds.The whole working mechanism may be thought of as a circular process, with MPO as its starting point [3].MPO undergoes a transition to an intermediate state, a designated molecule when interacting with H₂O₂.This is where the cycle diverges, with MPO primarily producing HOCl with the help of halides.In contrast, when H₂O₂ is abundant, MPO takes a different route [4,5].This alternative pathway involves numerous steps and different chemicals to achieve its result.When MPO takes in an additional electron, it becomes inactive, adding another degree of complication to the situation.However, there are redemptive routes provided by nature; inactive MPO may be converted back into its active form via interactions with oxygen and other mechanisms.In conclusion, MPO's capacity to adapt to surrounding molecules is crucial to its function in guarding the body against potential hazards [ 6,7,8].The amino acid taurine, or 2aminomethane-sulfonic acid, plays a crucial role in biology.The Latin word "Taurus," from which the name is derived, gives some idea of the historical background of its discovery.It's a sulfur-rich amino acid found in high concentrations in marine organisms and other animal tissues.On the other hand, its concentration in plants and fungal sources is still rather low.Muscles, the brain, the liver, and the kidneys all contain significant amounts of taurine.Taurine serves a wide variety of vital functions.It mostly contributes to the development of the functions of skeletal muscles, cardiovascular systems, central nervous systems, and the retina.
Studies have shown that not getting enough taurine might lead to subpar performance and trigger certain physiological abnormalities.Nutritionally speaking, taurine is a must-have for humans, and it becomes even more so in situations when the liver is unable to produce enough of it on its own [9,10,11,8].
Taurine may interact with proteins on a molecular level, mostly because of its amine (NH3 + ) group.
Methionine metabolism, bile acid production, intracellular transport systems, and general Sulphur metabolism are just a few of the many metabolic pathways in which it plays a role.Methionine, a source of Sulphur, is the first step in the production of homocysteine, from which taurine may be synthesized in many species.This molecule binds to serine and triggers the production of cysteine.The production of taurine involves a series of oxidation reactions: cysteine is converted to cysteine sulfonic acid by the enzyme cysteine dioxygenase (CDO).In the next enzymatic processes, guided by cysteine sulfonate decarboxylase (CSD) and hypotaurine dehydrogenase, hypotaurine, and then taurine are formed [12, 13, and 14].Understanding the pace and type of enzyme-catalyzed processes is made possible via the study of enzyme kinetics.In most cases, a clear rectangular hyperbola is seen when graphing the starting velocity of an enzyme versus its substrate concentration.The increase in enzyme activity is attenuated and eventually reaches a plateau as the substrate concentration rises.In contrast, the reaction rate in uncatalyzed reactions continues increasing with increasing reactant concentration [15].An increase in substrate concentration causes a linear or first-order increase in the rate of an enzyme-catalyzed reaction at low substrate concentrations.In contrast, increasing the concentration of the substrate does not significantly increase the rate of the enzyme-catalyzed reaction at extremely high concentrations.Zero-order kinetics describes this situation.It seems that the substrate concentration has little effect on the rate of this reaction.The Michaelis-Menten model, named after the eminent German scientist Leonor Michalis [16,17,18] is one of the most respected frameworks in the study of enzyme kinetics.An equation detailing the relationship between reaction velocity (V) and substrate concentration (S) is illustrative of this concept.Two key elements are highlighted in the well-known Michaelis-Menten equation: the Michaelis constant (Km), which represents the enzyme's affinity towards its substrate, and the maximum rate (Vmax) obtained by the system at maximal substrate saturation.The substrate concentration at which the response velocity (Vmax) is 50% of its highest value (Km) is particularly relevant.It is interesting to note that Km is invariant concerning enzyme concentration.In this framework, the rate of conversion of substrate to product is denoted by the velocity (v), which is often expressed as a mole per minute (mM) of product.
Assay Procedure: 1. Combine 0.45 mL of sample with 0.45 mL of Reagent 2 solution and mix thoroughly.
4. Follow the steps detailed in Table 1.V: is the reaction velocity.
Vmax: is the maximum reaction velocity.Km: is the Michaelis constant (substrate concentration at half-maximal reaction velocity) [S]: is the substrate concentration [16].

Assay Procedure with Taurine Inhibition:
The assay procedure mirrors the earlier steps with the addition of a 0.3 mM concentration of the taurine inhibitor.This concentration was chosen based on its efficacy as observed in preliminary results.For detailed steps, refer to the assay procedure section above.

Validation & Controls:
1-Negative controls (without substrate) were used to ensure specificity.2-Positive controls from known MPO activity samples were used for assay accuracy.
3-Triplicates were performed for each sample to ensure reproducibility.4-Inter-day and intra-day variations were measured for assay precision.

Results and Discussion:
Sample period: During the examination, 90 patients were studied.These samples included 30 people with type 2 diabetes, 30 were pre-diabetic subjects, and 30 were age-and gender-matched healthy controls (Figure 1).
Activity, MPO, HbA1c, BMI, age, and gender are compared between control, pre-diabetic, and diabetic groups in the table.The study analyses data using mean ± SD for continuous variables and frequencies and percentages for categorical variables (gender).The P-values show the statistical significance of the three groups' differences.
Activity There is a steady rise in mean activity MPO from the control group to the diabetes group The Pvalue (p <0.001) shows a significant difference between them and the activity there is a steady rise in mean activity MPO from the control group to the prediabetes group The P-value (p = 0.0002*) of shows a significant difference between them.The mean HbA1c level was found to grow gradually from the control group to the diabetes group (%).
Since HbA1c is a marker of long-term blood glucose management, the P-value of <0.001suggests a very significant difference between the three groups.The mean BMI rose significantly from the control to the diabetes group, with a P-value of <0.001.A higher BMI may lead to pre-diabetes and diabetes.The mean age rises somewhat from the control group to the diabetes group, although the change is not statistically significant (P-value = 0.2NS).
The asterisk (*) adjacent to P-values shows significant differences (p<0.05)across the three groups for activity MPO, HbA1c, and BMI, but not for age or gender.
double reciprocal plot) is a graphical representation of the Lineweaver-Burk equation for enzyme kinetics used in biochemistry.This graph was developed in 1934 by Hans Lineweaver and Dean Burk to compare the starting reaction rate (VO) to the substrate concentration [S].The hyperbolic curve has a very little slope at large substrate concentrations, making it difficult to identify the Vmax accomplishment.The Lineweaver-Burk plot, however, is a linear graph that results from contrasting 1/V with 1/[S].Extracting data like Km and Vmax and understanding the mechanism of enzyme inhibitors is a breeze with the help of this visual aid [19].Type 2 diabetes mellitus is becoming increasingly common, and it is a chronic, debilitating condition that has serious consequences [20].A combination of decreased pancreatic insulin production and the development of resistance to the activities of insulin on its target tissues leads to the onset of type 2 diabetes (T2D), a complicated metabolic condition.The accumulation of ectopic fat in vital organs such as the liver, pancreas, and skeletal muscles is central to the pathogenesis of T2D.Insulin resistance in these organs and the dysfunction of pancreatic beta cells are both exacerbated by the buildup of fat.Hyperglycemia (high blood sugar) is a complication that occurs in people with T2D [21, 22, and 23].The gradual development of T2D is a defining feature of the disease.Oftentimes, the typical signs of diabetes are ignored because the steady increase of hyperglycemia in the early stages is not severe enough.Therefore, many people go undetected for a long time.Undiagnosed people have an increased risk for both macrovascular and microvascular problems, even if they do not have any noticeable symptoms.In addition, some people with T2D may have normal or even increased insulin levels, but their raised blood glucose concentrations imply that their insulin values would be much higher if their beta cells were functioning normally.The failure of insulin production to adequately overcome insulin resistance is shown by this difference.While it is possible to improve insulin sensitivity with measures like weight reduction and medication, this improvement is usually only temporary [24, 25, 26].The possibility of developing T2D is increased by several risk factors.Age, excessive weight, and lack of physical activity are just a few examples.T2D is more common in women who have had gestational diabetes mellitus (GDM).High blood pressure, abnormal lipid profiles, and a strong hereditary propensity, particularly in close relatives, are additional risk factors.It's worth noting that, unlike type 1 diabetes, the genetics underlying T2D are still poorly understood.It is critical to do antibody testing to rule out a diagnosis of type 1 diabetes in people who appear with abnormal risk profiles for T2D or who develop the illness at a younger age [27, 28].Myeloperoxidase (MPO) enzymatic activity in the sera of pre-diabetic and type 2 diabetic persons will be measured using state-of-the-art spectroscopic methods.On top of that, we use cutting-edge kinetic modeling to figure out what these two factors do to MPO's Michaelis constant (Km) and maximal rate of enzymatic reaction (Vmax).Focusing on Km and Vmax, this study aims to determine how the addition of taurine affects these kinetic parameters of MPO from a molecular dynamics standpoint.As new data suggests that taurine has a function in glucose metabolism, it seemed a natural choice.Materials and Methods This case-control study was conducted in the Department of Chemistry, Biochemistry, and Physiology, Iraqi University College of Medicine.The study was executed during the term from December 2022 to March 2023.The Ethics Committee of the College of Medicine at Iraqi University duly approved the research protocol for this study, in compliance with the stipulations outlined in the 2013 Helsinki Declaration.All study participants provided informed consent before their involvement.

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Km and Vmax Calculation:Utilize the Lineweaver-Burk plot.The X-axis intercept indicates -1/Km, and the Y-axis intercept represents 1/Vmax.
Figure1 : Pie-charts showing the distribution of the studied groups.

- Quantification of MPO Activity:
 MPO Activity is in units per liter (U/L).ΔA represents the change in absorbance.Total Is the total volume.Is the sample volume.Is a factor.b, V₁, V₂ are constants.29 -

Table 1 Assay Procedure
To ascertain the Km and Vmax of MPO in patient sera, we utilize five varying substrate concentrations of H₂O₂.We aim to ascertain the Km and Vmax of MPO in patient sera.The Lineweaver-Burk plot, derived from the Michaelis-Menten equation, is used.It's acknowledged that the Lineweaver-Burk plot can distort data due to transformation; thus, data integrity will be ensured by repeated measurements.