Kinetic and Structural Evidences on Human Prolidase Pathological Mutants Suggest Strategies for Enzyme Functional Rescue

Abstract

Prolidase is the only human enzyme responsible for the digestion of iminodipeptides containing proline or hydroxyproline at their C-terminal end, being a key player in extracellular matrix remodeling. Prolidase deficiency (PD) is an intractable loss of function disease, characterized by mutations in the prolidase gene. The exact causes of activity impairment in mutant prolidase are still unknown. We generated three recombinant prolidase forms, hRecProl-231delY, hRecProl-E412K and hRecProl-G448R, reproducing three mutations identified in homozygous PD patients. The enzymes showed very low catalytic efficiency, thermal instability and changes in protein conformation. No variation of Mn(II) cofactor affinity was detected for hRecProl-E412K; a compromised ability to bind the cofactor was found in hRecProl-231delY and Mn(II) was totally absent in hRecProl-G448R. Furthermore, local structure perturbations for all three mutants were predicted by in silico analysis. Our biochemical investigation of the three causative alleles identified in perturbed folding/instability, and in consequent partial prolidase degradation, the main reasons for enzyme inactivity. Based on the above considerations we were able to rescue part of the prolidase activity in patients’ fibroblasts through the induction of Heath Shock Proteins expression, hinting at new promising avenues for PD treatment.

Substrate specificity and reaction mechanism of human prolidase

“…As a consequence of this, the nucleophilic attack would be facilitated. The proposed mechanism is also supported by kinetic analysis of the Glu412Lys mutant .…”

Structural basis for prolidase deficiency disease mechanisms

“…This change is accompanied by an increased active site flexibility and leads to a complete loss of enzyme activity. It is interesting to note for this variant, that in earlier modeling studies , this effect was not predicted. It is also surprising that biochemical studies on this variant mutant suggested the presence of both ions and hinted at slightly increased affinity .…”

“…It is interesting to note for this variant, that in earlier modeling studies , this effect was not predicted. It is also surprising that biochemical studies on this variant mutant suggested the presence of both ions and hinted at slightly increased affinity . This is clearly in contradiction to the results presented here.…”

“…For some time, researchers have tried to explain the influence of particular mutations on the protein structure and function and their relation to disease. Due to the lack of experimental evidence for the structural differences induced by single amino acid alterations, molecular modeling was employed as the method of choice to investigate this problem . Here, we present for the first time direct evidence for the structural changes induced by the relevant point mutations.…”

Variation of Prolidase Activity: A Predictor of Neuropathy due to Diabetes Mellitus?

“…Prolidase (PLD, EC 3.4.13.9) is a member of the matrix metalloproteinase family (5,6) . It is the only human enzyme responsible for the digestion of iminodipeptides containing proline or hydroxyproline at their C-terminal end, being a key player in collagen turnover, extracellular matrix remodeling and cell growth (5,(7)(8)(9) . Uzar et al (2012) stated that there is increased plasma prolidase enzyme activity in neuropathy due to diabetes (DN) as evidence of increased collagen turnover (10) .…”