Evidence for the involvement of tyrosine-69 in the control of stereospecificity of porcine pancreatic phospholipase A2

We have studied the role of Tyr-69 of porcine pancreatic phospholipase A2 in catalysis and substrate binding, using site-directed mutagenesis. A mutant was constructed containing Phe at position 69. Kinetic characterization revealed that the Phe-69 mutant has retained enzymatic activity on monomeric and micellar substrates, and that the mutation has only minor effects on kcat and Km. This shows that Tyr-69 plays no role in the true catalytic events during substrate hydrolysis. In contrast, the mutation has a profound influence on the stereospecificity of the enzyme. Whereas the wild-type phospholipase A2 is only able to catalyse the degradation of sn-3 phospholipids, the Phe-69 mutant hydrolyses both the sn-3 isomers and, at a low (1-2%) rate, the sn-1 isomers. Despite the fact that the stereospecificity of the mutant phospholipase has been altered, Phe-69 phospholipase still requires Ca2+ ions as a cofactor and also retains its specificity for the sn-2 ester bond. Our data suggest that in porcine pancreatic phospholipase A2 the hydroxyl group of Tyr-69 serves to fix and orient the phosphate group of phospholipid monomers by hydrogen bonding. Because no such interaction can occur between the Phe-69 side-chain and the phosphate moiety of the substrate monomer, the mutant enzyme loses part of its stereospecificity but not its positional specificity

Comparison of Functional Outcome Scores in Radial Polydactyly

Background: A wide range of outcome assessment systems have been used to describe the results and evaluate residual impairment after surgery for radial polydactyly. We conducted a study to determine which of these assessment systems should be considered superior for the most common types of radial polydactyly (types ll and IV). Methods: Ten outcome assessment systems were selected. Three examiners independently evaluated thirty-seven patients, aged four to twenty-two years, with radial polydactyly. Patients completed two manual activity questionnaires. lnterobserver reliability was determined with use of an intraclass correlation coefficient (ICC). Validity was assessed by correlating the results derived with the outcome assessment systems with functional visual analog scale (VAS), aesthetic VAS, and manual activity questionnaire scores. Results: Thirty-seven patients (forty-one hands with radial polydactyly) were evaluated. All patients were assessed by at least two examiners. Reliability was highest for the Japanese Society for Surgery of the Hand (JSSH), Cheng et al., and Tada et al. assessment systems (overall ICCs >= 0.70). The JSSH system had the highest overall correlations (r(s) ranging from 0.48 to 0.80 and 0.45 to 0.63) with functional and aesthetic VAS scores. No significant correlations were found between the outcome scores and the results of the manual activity questionnaires after an average follow-up time of 112 months. Conclusions: Interobserver reliability was highest for the JSSH classification, which also showed superior correlations with both examiner-rated and patient-rated VAS scores for functional and aesthetic outcome compared with the other nine assessment systems. The finding of a poor correlation between the outcome scores and the results of manual activity questionnaires is in agreement with findings in published literature. We recommend the JSSH assessment method for the scientific evaluation of the outcomes, in terms of body structure and function, of the treatment of radial polydactyly

A Multicenter Comparative Study of Two Classification Systems for Radial Polydactyly

Background: The aim of this study was to compare type occurrence and reliability of the Wassel and Rotterdam classifications for radial polydactyly. Methods: The authors classified a large population of radial polydactyly patients from two European clinics using both classification systems, and compared the incidences of the different types to a population derived from a systematic literature review. The authors further assessed intraobserver and interobserver reliability of both classification systems in a test-retest design with seven observers, using kappa statistics. Results: Forty percent of the 520 cases with available radiographs could not be classified using the Wassel classification, whereas all cases could be classified using the Rotterdam classification. All unclassifiable cases had aberrant components; the majority were of the triphalangeal (63 percent), deviating (43 percent), or hypoplastic (39 percent) kind. Types III, IV, and VI occurred more often when using the Rotterdam classification. Intraobserver and interobserver reliability was comparable for both classification systems ( = 0.87 versus = 0.83, and = 0.65 versus = 0.70). Types II and IV had the lowest reliability in both the Wassel and Rotterdam classifications ( = 0.30 to 0.59). Aberrant components indicating deviation and hypoplasia had the lowest reliability in the Rotterdam classification ( = 0.19 to 0.45). Conclusions: The Rotterdam classification has broader classification possibilities and similar intraobserver and interobserver reliability compared with the Wassel classification. Although it is more complex and the aberrant components should be more strictly defined to increase its clinical relevance, we recommend using the Rotterdam classification. CLINICAL QUESTION/LEVEL OF EVIDENCE: Diagnostic, I

A clinically weighted approach to outcome assessment in radial polydactyly

Currently available outcome assessment systems for radial polydactyly are mainly based on expert opinion. The aim of this study was to develop an outcome assessment system based on clinical data. We performed linear regression analysis on data from a multicentre study of 121 patients with radial polydactyly types II, IV and VII to develop a clinically weighted outcome assessment system. Items were weighted according to their influence on overall functional and aesthetic outcome in the regression analysis. Active flexion, scar appearance and prominence at amputation site were the main items influencing overall functional and aesthetic outcome (beta = 0.393, beta = 0.326 and beta = 0.288, respectively). Palmar abduction, metacarpophalangeal joint deviation and nail appearance influenced overall functional and aesthetic outcome the least (beta = -0.002, beta = -0.104 and beta = 0.070, respectively). Our proposed assessment system for radial polydactyly reflects the way clinicians value individual aspects of outcome as determinants of overall outcome and helps guide future treatment and evaluation of outcome

Influence of size and polarity of residue 31 in porcine pancreatic phospholipase A2 on catalytic properties

Residue 31 of porcine pancreatic phospholipase A2 (PLA2) is located at the entrance to the active site. To study the role of residue 31 in PLA2, six mutant enzymes were produced by site-directed mutagenesis, replacing Leu by either Trp, Arg, Ala, Thr, Ser or Gly. Direct binding studies indicated a three to six times greater affinity of the Trp31 PLA2 for both monomeric and micellar substrate analogs, relative to the wild-type enzyme. The other five mutants possess an unchanged affinity for monomers of the product analog n-decylphosphocholine and for micelles of the diacyl substrate analog rac-1,2-dioctanoylamino-dideoxy-glycero-3-phosphocholine. The affinities for micelles of the monoacyl product analog n-hexadecylphosphocholine were decreased 9-20 times for these five mutants. Kinetic studies with monomeric substrates showed that the mutants have Vmax values which range between 15 and 70% relative to the wild-type enzyme. The Vmax values for micelles of the zwitterionic substrate 1,2-dioctanoyl-sn-glycero-3-phosphocholine were lowered 3-50 times. The Km values for the monomeric substrate and the Km values for the micellar substrate were hardly affected in the case of five of the six mutants, but were considerably decreased when Trp was present at position 31. The results of these investigations point to a versatile role for the residue at position 31: involvement in the binding and orientating of monomeric substrate (analogs), involvement in the binding of the enzyme to micellar substrate analogs and possibly involvement in shielding the active site from excess water.