Catalytic role of the active site histidine of porcine pancreatic phospholipase A2 probed by the variants H48Q, H48N and H48K

The catalytic contribution of His48 in the active site of porcinepancreatic phospholipase A2 was examined using site-directedmutagenesis. Replacement of His48 by lysine (H48K) gives riseto a protein having a distorted lipid binding pocket. Activityof this variant drops below the detection limit which is 10⁷-foldlower than that of the wild-type enzyme. On the other hand,the presence of glutamine (H48Q) or asparagine (H48N) at thisposition does not affect the structural integrity of the enzymeas can be derived from the preserved lipid binding propertiesof these variants. However, the substitutions H48Q and H48Nstrongly reduce the turnover number, i.e. by a factor of 10⁵.Residual activity is totally lost after addition of a competitiveinhibitor. We conclude that proper lipid binding on its ownaccelerates ester bond hydrolysis by a factor of 10². With theselected variants, we were also able to dissect the contributionof the hydrogen bond between Asp99 and His48 on conformationalstability, being 5.2 kJ/mol. Another hydrogen bond with His48is formed when the competitive inhibitor (R)-2-dodecanoylamino-hexanol-1-phosphoglycolinteracts with the enzyme. Its contribution to binding of theinhibitor in the presence of an interface was found to be 5.7kJ/mol.     

Incorporation of an unnatural amino acid in the active site of porcine pancreatic phospholipase A2. Substitution of histidine by l,2,4-triazole-3-alanine yields an enzyme with high activity at acidic pH

The effect of the substitution of the active site histidine48 by the unnatural 1,2,4-triazole-3-alanine (TAA) amino acidanalogue in porcine pancreas phospholipase A₂ (PLA₂) was studied.TAA was introduced biosynthetically using a his-auxotrophicEscherichia coli strain. To study solely the effect of the substitutionof the active site histidine, two nonessential histidines (i.e.His17 and His 115) were replaced by asparagines, resulting ina fully active mutant enzyme (His-PLA₂). In this His-PLA₂ thesingle histidine at position 48 was substituted by TAA withan incorporation efficiency of about 90%, giving a mixture ofHis-PLA₂ and TAA-PLA₂. Based on the charge difference at acidicpH, both forms could be separated by FPLC, allowing for thepurification of TAA-PLA₂ free from His-PLA₂. At pH 6, TAA-PLA₂has a fivefold reduced activity compared with His-PLA₂. Thisreduced activity paralells a reduced rate of covalent modificationwith p-nitrophenacyl bromide of TAA-PLA₂ compared with His-PLA₂.Competitive inhibition gave comparable IC₅₀ values for WT-PLA₂,His-PLA₂ and TAA-PLA₂. These results indicate that the reductionin activity is not caused by a different affinity for the substrate,but more likely results from a reduced k_cat value in TAA-PLA₂.The enzymatic activities for native and mutant PLA₂s were measuredat different pH values. For WT-PLA₂ and His-PLA₂ the activityis optimal at pH 6 and is strongly deminished at acidic pH,with no observable activity at pH 3. In contrast, TAA-PLA₂ isas active at pH 3 as at pH 6. Most likely, the decrease in activityobserved for WT-PLA₂ and His-PLA₂ is caused by the protonationof the active site His48, which is the general base involvedin the activation of the nucleophilic water molecule. In TAA-PLA₂,however, the active site residue TAA48 is unprotonated at bothpH 3 and 6 as a result of the low pK_a of TAA compared with histidine.     

Management practices associated with low, medium, and high somatic cell counts in bulk milk

Management practices associated with bulk milk somatic cell counts (SCC) were studied for 201 dairy herds grouped into three categories according to bulk milk SCC. The cumulative production of fat-corrected milk over 305 d of lactation and category for bulk milk SCC were highly correlated; herds within the low category had the highest milk production. Differences in bulk milk SCC among the categories were well explained by the management practices studied. This correlation was not only true for the difference between the high (250,000 to 400,000) and low (< or = 150,000) categories for bulk milk SCC but also for the difference between the medium (150,000 to 250,000) and low categories and the high and medium categories. Management practices that are known to be important for herds in the high category for bulk milk SCC, such as dry cow treatment, milking technique, postmilking teat disinfection, and antibiotic treatment of clinical mastitis, were also found to be important in the explanation of the difference between herds in the medium and low categories for bulk milk SCC. More attention was paid to hygiene for herds in the low category than for herds in the medium or high category. Supplementation of the diet with minerals occurred more frequently for cows in the low category for bulk milk SCC than for cows in the medium and high categories.     

Management practices associated with the incidence rate of clinical mastitis.

Risk factors for the incidence rate of clinical mastitis were studied in 274 Dutch dairy herds. Variables that were associated with resistance to disease were the feeding, housing, and milking machine factors. Variables that were associated with exposure were grazing, combined housing of dry cows and heifers, and calving area hygiene. Postmilking teat disinfection in herds with a low bulk milk somatic cell count and years of practicing dry cow therapy were positively associated with the incidence rate of clinical mastitis. Herds with a low bulk milk somatic cell count and in which postmilking teat disinfection was not used had lower incidence rates of clinical mastitis than did other herds. The incidence rate of clinical mastitis caused by Escherichia coli was mostly related to housing conditions, hygiene, and machine milking. The incidence rate of clinical mastitis caused by Staphylococcus aureus was mostly related to factors associated with bulk milk somatic cell count and factors that might be due to cause and effect reversal. A strong positive correlation existed between the incidence rate of clinical mastitis caused by Streptococcus dysgalactiae and the incidence rate of clinical mastitis caused by Staph. aureus. The incidence rate of clinical mastitis caused by Streptococcus dysgalactiae was related to nutrition, milking technique, and machine milking. The incidence rate of clinical mastitis caused by Streptococcus uberis was associated with factors related to housing, nutrition, and machine milking.